0013-7227/78/1035-1888$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 5 Printed in U.S.A.
Effects of Intraventricular Injection of y-Aminobutyric Acid (GABA) on Plasma Growth Hormone and Thyrotropin in Conscious Ovariectomized Rats* E. VIJAYANf AND S. M. McCANN Department of Physiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 ABSTRACT. Control injections of saline ip or into the third ventricle (2 fil) failed to modify concentrations of GH or TSH in plasma of conscious, free-moving, ovariectomized (OVX) or OVX, estrogen- and progesteronetreated (OEP) rats. Intraventricular injection of y-aminobutyric acid (GABA) in doses ranging from 0.1-8 fimol induced a rapid elevation in plasma GH within 10 min of injection which was dose related and the elevation persisted for the 60-min duration of the study in both OVX and OEP rats. This effect was completely blocked by the GABA antagonist, bicuculline, which by itself had no effect on plasma GH in doses of 1 or 2 /imol ip. By contrast, GABA administration lowered plasma TSH, which became apparent within 10 min in OVX rats and persisted for the duration of the experiment
f I ^HERE is increasing evidence that y-ami-L nobutyric acid (GABA) may function as a neurotransmitter in the mammalian central nervous system (CNS), and this has led to studies of its possible influence on pituitary hormone secretion (1,2). Thus, ACTH release in response to surgical trauma was blocked by intraventricular infusion of GABA and was potentiated by GABA antagonists (3). Intraventricular injection of GABA into male rats (4) and conscious, ovariectomized (OVX) or OVX, estrogen- and progesterone-primed (OEP) rats (5) induced LH but not FSH release. In the case of PRL, both stimulatory and inhibitory effects have been reported (6-11) and in a more recent study (5), intraventricular injection of low doses of GABA
and appeared to be dose related at the initial sampling time of 10 min. The inhibitory effect of GABA was less prominent in OEP rats. Bicuculline, which by itself had no effect on plasma TSH, completely blocked the inhibitory effect of GABA in OVX rats. The effects of GABA were mediated centrally since 4 /xmol GABA given iv or in vitro incubation of hemipituitaries with varying doses of GABA had no effect on either hormone. It is suggested that GABA can stimulate release of GH by a hypothalamic action, whereas it can inhibit the release of TSH also by an action on the hypothalamus. Since bicuculline did not modify the levels of GH or TSH, it is not clear whether GABA has a physiological role in control of the two hormones. (Endocrinology 103:1888, 1978)
lowered PRL whereas higher doses elevated the levels of the trophin, suggesting that differences in dosage could have accounted for the earlier discrepancies. The purpose of the present investigation was to evaluate the effects of injection of GABA into the third ventricle on plasma GH and TSH levels in unanesthetized, OVX rats. Ovariectomized rats primed with estrogen and progesterone were also used to determine if the responses were influenced by the steroid hormone environment. Materials and Methods
Virgin female Sprague-Dawley rats (Simonsen Laboratory, Gilroy, CA), weighing 200-220 g, were housed under controlled conditions of light and temperature (lights on 0500-1900 h) with free access to Purina rat chow and water. One week after Received March 18,1978. Address requests for reprints to: Dr. S. M. McCann, arrival, they were OVX under light ether anestheDepartment of Physiology, University of Texas Health sia. Three to four weeks after OVX, the animals Science Center, Southwestern Medical School, 5323 were used for the experiments. In some experiments Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by grants from NIH (AM- OVX, OEP rats were also used to determine if the steroid treatment influenced the responsiveness of 10073 and HD-09988) and the Ford Foundation. f On leave from Delhi University, India. GH- and TSH-releasing mechanisms to intraven1888
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:30 For personal use only. No other uses without permission. . All rights reserved.
GABA EFFECT ON GH AND TSH
1889
tricular GABA. The rats were primed with estradiol until the day of the assay. To determine if the effects of these doses of benzoate (50 jug sc) and progesterone (25 mg sc) 72 intraventricular GABA were mediated centrally, its h before use (12). effects after iv pulse injection and from hemipituitaries incubated in vitro were also evaluated. In Experimental procedure vitro incubation of hemipituitaries from OVX rats Four to six days before the experiment, a 23- in tissue culture medium 199 (Difco Laboratories) gauge stainless steel cannula (17 mm in length) was with varying doses of GABA was performed as implanted into the third ventricle of the experimen- described before (15). At the termination of incutal rats, as previously described by Antunes-Ro- bation, media were removed and kept frozen for drigues and McCann (13). Each cannula was pro- later RIA of GH and TSH. vided with a mandril to prevent its obstruction. TSH concentration in the plasma and incubation The correct location of the cannula in the third medium were determined by the NIAMDD RIA ventricle was confirmed when cerebrospinal fluid kit for rat TSH. The concentrations of TSH are flowed continuously from the cannula after removal expressed in terms of the RP-1 standard provided. of the mandril. After surgery, the animals were The lower level of sensitivity of the assay was 32 returned to their own individual cages until the day ng/ml. All samples belonging to each individual of the experiment. Indwelling catheters were intro- experimental group were always determined in the duced into the external jugular vein 24 h before the same assay. GH concentrations were also deterexperiment, using the technique of Harms and mined by the NIAMDD kit for GH RIA. The Ojeda (14). On the day of the experiment, an exten- results are expressed in terms of the NIAMDD-rat sion of polyethylene tubing (PE50; 12 in. in length) GH-RP-1 standard provided with the kit. All samfilled with heparin-0.9% NaCl was attached to the ples for both hormones were measured in duplicate distal end of the jugular cannula and the animals and at two different dilutions. The quality of the were left undisturbed for 30-60 min. During this assays was controlled according to the criteria protime, a preinjection blood sample (0.6-0.8 ml) was posed by Rodbard et al. (16). withdrawn over a period of 60 sec. Microinjections of saline or GABA (Sigma Statistics Chemicals, lot 32C-2210) were performed according Significance of differences between pre- and postto the following procedure. Test material was injection plasma TSH and GH levels in the same freshly prepared in 0.9% NACl adjusted to pH 7.2 and injected within 30 min of preparation. Injec- group was calculated by the paired t test. Signifitions were made in a volume of 2 /xl using a 10-jd cance of differences between groups were deterHamilton microsyringe connected by polyethylene mined by using one-way analysis of variance, foltubing (PE10) to a 30-gauge stainless steel cannula, lowed by Scheffe's test for multiple comparisons. which fit within and extended to the lower tip of the permanent cannula. The tubing and the cannula had been previously filled with the solution to Results be injected. To evaluate the effects of a GABA antagonist, Initial hormone levels OVX rats were pretreated with either 1 or 2 /umol Initial levels of GH were readily detectable bicuculline (Sigma Chemicals, lot 115C-0291; dissolved in 0.1 ml ethyl acetate) or equal volume of in the OVX animals and they were not signifdiluent injected ip either alone or followed 15 min icantly altered by treatment with estrogen and later by third ventricular injection of 4 /imol GABA. progesterone (Figs. 1-7). Similarly, the readily In all cases, intraventricular injection time was detectable TSH levels were not altered by the about 60 sec and all experiments were performed in steroid pretreatment. the morning between 0900-1100 h. Heparinized blood samples (0.6-0.8 ml) were collected from the external jugular vein cannula at Control injections varying intervals (see Results) while the animal Plasma GH and TSH levels were not altered was freely moving in the cage. The volume of all by intraventricular injection of 2 jul isotonic samples was replaced immediately after each bleedsaline in OVX and OEP rats or by ip injection ing by an equivalent volume of saline. Plasma was separated by centrifugation at 4 C and stored frozen of 0.1 ml saline in OVX animals (Figs. 1-7).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:30 For personal use only. No other uses without permission. . All rights reserved.
VIJAYAN AND McCANN
1890
E1OO
I
E2 IE1 E! • E3
Sollrta O.ipmol 2 jimol 4 />mol 8 />mol
•p
Vol. 103, No. 5 Printed in U.S.A.
Effects of Intraventricular Injection of y-Aminobutyric Acid (GABA) on Plasma Growth Hormone and Thyrotropin in Conscious Ovariectomized Rats* E. VIJAYANf AND S. M. McCANN Department of Physiology, University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 ABSTRACT. Control injections of saline ip or into the third ventricle (2 fil) failed to modify concentrations of GH or TSH in plasma of conscious, free-moving, ovariectomized (OVX) or OVX, estrogen- and progesteronetreated (OEP) rats. Intraventricular injection of y-aminobutyric acid (GABA) in doses ranging from 0.1-8 fimol induced a rapid elevation in plasma GH within 10 min of injection which was dose related and the elevation persisted for the 60-min duration of the study in both OVX and OEP rats. This effect was completely blocked by the GABA antagonist, bicuculline, which by itself had no effect on plasma GH in doses of 1 or 2 /imol ip. By contrast, GABA administration lowered plasma TSH, which became apparent within 10 min in OVX rats and persisted for the duration of the experiment
f I ^HERE is increasing evidence that y-ami-L nobutyric acid (GABA) may function as a neurotransmitter in the mammalian central nervous system (CNS), and this has led to studies of its possible influence on pituitary hormone secretion (1,2). Thus, ACTH release in response to surgical trauma was blocked by intraventricular infusion of GABA and was potentiated by GABA antagonists (3). Intraventricular injection of GABA into male rats (4) and conscious, ovariectomized (OVX) or OVX, estrogen- and progesterone-primed (OEP) rats (5) induced LH but not FSH release. In the case of PRL, both stimulatory and inhibitory effects have been reported (6-11) and in a more recent study (5), intraventricular injection of low doses of GABA
and appeared to be dose related at the initial sampling time of 10 min. The inhibitory effect of GABA was less prominent in OEP rats. Bicuculline, which by itself had no effect on plasma TSH, completely blocked the inhibitory effect of GABA in OVX rats. The effects of GABA were mediated centrally since 4 /xmol GABA given iv or in vitro incubation of hemipituitaries with varying doses of GABA had no effect on either hormone. It is suggested that GABA can stimulate release of GH by a hypothalamic action, whereas it can inhibit the release of TSH also by an action on the hypothalamus. Since bicuculline did not modify the levels of GH or TSH, it is not clear whether GABA has a physiological role in control of the two hormones. (Endocrinology 103:1888, 1978)
lowered PRL whereas higher doses elevated the levels of the trophin, suggesting that differences in dosage could have accounted for the earlier discrepancies. The purpose of the present investigation was to evaluate the effects of injection of GABA into the third ventricle on plasma GH and TSH levels in unanesthetized, OVX rats. Ovariectomized rats primed with estrogen and progesterone were also used to determine if the responses were influenced by the steroid hormone environment. Materials and Methods
Virgin female Sprague-Dawley rats (Simonsen Laboratory, Gilroy, CA), weighing 200-220 g, were housed under controlled conditions of light and temperature (lights on 0500-1900 h) with free access to Purina rat chow and water. One week after Received March 18,1978. Address requests for reprints to: Dr. S. M. McCann, arrival, they were OVX under light ether anestheDepartment of Physiology, University of Texas Health sia. Three to four weeks after OVX, the animals Science Center, Southwestern Medical School, 5323 were used for the experiments. In some experiments Harry Hines Boulevard, Dallas, Texas 75235. * This work was supported by grants from NIH (AM- OVX, OEP rats were also used to determine if the steroid treatment influenced the responsiveness of 10073 and HD-09988) and the Ford Foundation. f On leave from Delhi University, India. GH- and TSH-releasing mechanisms to intraven1888
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:30 For personal use only. No other uses without permission. . All rights reserved.
GABA EFFECT ON GH AND TSH
1889
tricular GABA. The rats were primed with estradiol until the day of the assay. To determine if the effects of these doses of benzoate (50 jug sc) and progesterone (25 mg sc) 72 intraventricular GABA were mediated centrally, its h before use (12). effects after iv pulse injection and from hemipituitaries incubated in vitro were also evaluated. In Experimental procedure vitro incubation of hemipituitaries from OVX rats Four to six days before the experiment, a 23- in tissue culture medium 199 (Difco Laboratories) gauge stainless steel cannula (17 mm in length) was with varying doses of GABA was performed as implanted into the third ventricle of the experimen- described before (15). At the termination of incutal rats, as previously described by Antunes-Ro- bation, media were removed and kept frozen for drigues and McCann (13). Each cannula was pro- later RIA of GH and TSH. vided with a mandril to prevent its obstruction. TSH concentration in the plasma and incubation The correct location of the cannula in the third medium were determined by the NIAMDD RIA ventricle was confirmed when cerebrospinal fluid kit for rat TSH. The concentrations of TSH are flowed continuously from the cannula after removal expressed in terms of the RP-1 standard provided. of the mandril. After surgery, the animals were The lower level of sensitivity of the assay was 32 returned to their own individual cages until the day ng/ml. All samples belonging to each individual of the experiment. Indwelling catheters were intro- experimental group were always determined in the duced into the external jugular vein 24 h before the same assay. GH concentrations were also deterexperiment, using the technique of Harms and mined by the NIAMDD kit for GH RIA. The Ojeda (14). On the day of the experiment, an exten- results are expressed in terms of the NIAMDD-rat sion of polyethylene tubing (PE50; 12 in. in length) GH-RP-1 standard provided with the kit. All samfilled with heparin-0.9% NaCl was attached to the ples for both hormones were measured in duplicate distal end of the jugular cannula and the animals and at two different dilutions. The quality of the were left undisturbed for 30-60 min. During this assays was controlled according to the criteria protime, a preinjection blood sample (0.6-0.8 ml) was posed by Rodbard et al. (16). withdrawn over a period of 60 sec. Microinjections of saline or GABA (Sigma Statistics Chemicals, lot 32C-2210) were performed according Significance of differences between pre- and postto the following procedure. Test material was injection plasma TSH and GH levels in the same freshly prepared in 0.9% NACl adjusted to pH 7.2 and injected within 30 min of preparation. Injec- group was calculated by the paired t test. Signifitions were made in a volume of 2 /xl using a 10-jd cance of differences between groups were deterHamilton microsyringe connected by polyethylene mined by using one-way analysis of variance, foltubing (PE10) to a 30-gauge stainless steel cannula, lowed by Scheffe's test for multiple comparisons. which fit within and extended to the lower tip of the permanent cannula. The tubing and the cannula had been previously filled with the solution to Results be injected. To evaluate the effects of a GABA antagonist, Initial hormone levels OVX rats were pretreated with either 1 or 2 /umol Initial levels of GH were readily detectable bicuculline (Sigma Chemicals, lot 115C-0291; dissolved in 0.1 ml ethyl acetate) or equal volume of in the OVX animals and they were not signifdiluent injected ip either alone or followed 15 min icantly altered by treatment with estrogen and later by third ventricular injection of 4 /imol GABA. progesterone (Figs. 1-7). Similarly, the readily In all cases, intraventricular injection time was detectable TSH levels were not altered by the about 60 sec and all experiments were performed in steroid pretreatment. the morning between 0900-1100 h. Heparinized blood samples (0.6-0.8 ml) were collected from the external jugular vein cannula at Control injections varying intervals (see Results) while the animal Plasma GH and TSH levels were not altered was freely moving in the cage. The volume of all by intraventricular injection of 2 jul isotonic samples was replaced immediately after each bleedsaline in OVX and OEP rats or by ip injection ing by an equivalent volume of saline. Plasma was separated by centrifugation at 4 C and stored frozen of 0.1 ml saline in OVX animals (Figs. 1-7).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 05:30 For personal use only. No other uses without permission. . All rights reserved.
VIJAYAN AND McCANN
1890
E1OO
I
E2 IE1 E! • E3
Sollrta O.ipmol 2 jimol 4 />mol 8 />mol
•p
