- Email: [email protected]
Non-neuronal endogenous GABA efflux from the rat oviduct
L i f e Sciences, Vol. P r i n t e d in the U S A
52, pp.
811-818
Pergamon
Press
NON-NEURONAL ENDOGENOUS GABA EFFLUX FROM THE RAT OVIDUCT Maria In~s Forray, Patricia Hidalgo, Felipe Diaz and Jorge Belmar Laboratory of Biochemical Pharmacology, Dept. Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Alameda 340, P.O. Box 114-D, Santiago, Chile. (Received
in final
form D e c e m b e r
15,
1992)
Summary The subcellular distribution of Gamma-aminobutyric acid (GABA) was studied in the rat oviduct. The highest content of GABA was found in the soluble fraction. The effect of chemical stimulation of the endogenous GABA effiux from the rat Qviduct was examined. High K + concentrations could not induce elevation of the GABA efflux. Instead, a continuous spontaneous GABA effiux without change for long periods of time was observed. The total GABA content and GABA concentration were determined in the rat oviduct on days 1, 5, 10, 15, 30, 35 and 40 of the postnatal period and also during the estrous cycle. During postnatal development the GABA levels increase gradually with time reaching at prepuberal age a concentration similar to that found in diestrous rats. In the estrous cycle both GABA content and GABA concentration reached the highest value in the proestrous and the lowest value in the estrous phase. These findings support the hypothesis that GABA effiux may be modulated by the changes in oviductal fluid volume during the estrous cycle. Gamma-aminobutyric acid (GABA) is considered in the central nervous system (CNS). The presence of in the peripheral organs and tissues motivate current plays a neurotransmitter role or whether some cells synthesize G A B A (for review see 1,2,3).
the principal inhibitory neurotransmitter this amino acid and its wide distribution interest in establishing whether GABA of these tissues and organs themselves
The rat oviduct contains high GABA amounts (4,5). Also, this organ contains significant glutamic acid descarboxylase (GAD) activity, the rate-limiting enzyme in the synthesis of G A B A in the CNS (6,7). This enzyme has two forms GAD 65 and GAD 67 encoded by two different genes. It has been shown that GAD 65 is also present in the rat oviduct (8). On the other hand, it has been shown that GABA levels or GAD activity change in different endocrine situations like estrous cycle (4,5,9,10,11), pregnancy (11,12), prepuberal period (13) and with different endocrine manipulations like ovariectomy and hypophysectomy (14,15). GABA secretion from the oviducts and its fluctuating level during estrous cycle in the ovarian bursa fluid were described by Louzan et al. (1986). The possibility that GABA could mediate the neuronal regulation of endocrine activity in the oviduct has also been studied. These studies have shown that G A B A content or GAD activity change after denervation procedures, such as vagotomy, subcutaneous transplantation of the oviduct (6) and section of the vascular nerve bundles (7,15). However, biochemical studies have shown the presence of highly concentrated GABA and GAD activity in oviductal mucosa and in the muscle layer (14), suggesting a non-neuronal nature for oviductal GABA. This is further supported by immunohystochemical evidences showing the presence of GABA, GABA-transaminase and GAD in the epithelial cells of the mucosa. GABA exhibit a specific localization in the basal Copyright
0 0 2 4 - 3 2 0 5 / 9 3 $6.00 + .00 © 1993 P e r g a m o n Press Ltd All r i g h t s
reserved.
812
O v i d u c t a l F l u i d V o l u m e and G A B A E f f l u x
Vol.
52, No.
9, 1993
body of the cilia (16,17). The present study was performed to elucidate the characteristics of the oviductal G A B A compartment and the possible mechanisms involved in G A B A secretion.
Materials and Methods Animals: Female Sprague-Dawley rats weighing 180-250 g were used, otherwise specified. For the postnatal level experiments, animals on day 1, 5, 10, 30, 35 and 40 of age were used weighing 10 to 110 g. The stages of the estrous cycle were assessed by vaginal smears examination and only animals presenting two or more consecutive 4 days cycles were used.
Tissue samples: The animals were sacrified, ovaries and oviducts were quickly removed, put at 4-0C and cleaned of connective tissue and fat. Oviducts were homogenized in ice cold 0.2 N perchloric acid with a glass-glass homogenizer and centrifugated for 20 rain at 12000 xg at 4-°C. The clear supernatant fractions were analized for G A B A content.
Subcellular fraetionation: Oviducts were homogenized in 10 % (w/v) buffered 0.32 M sucrose (pH 7.4) with a glass-glass homogenizer. This homogenate was subjected to differential centrifugation. The following fractions were obtained: a crude nuclear pellet (P1) by centrifugation at 800 xg for 10 rain.; a crude mitochondrial pellet (P2) by centrifugation at 8500 xg for 20 rain.; a microsomal pellet (P3) and a soluble fraction ($3) by centrifugation at 130,000 xg for 90 rain. All fractions were collected and kept frozen at - 20-°C until G A B A content was determined.
Endogenous G A B A efflux: For each experiment both oviducts obtained from rats on estrous or diestrous phase were cut in 8 pieces each and incubated in Krebs Ringer Bicarbonate solution (KRB) as previously described (18). For high K÷-KRB, equimolar amounts of NaC1 were replaced by KCI to maintain an iso-osmotic condition. Tissue were put in a stainless steel basket and preincubated in KRB for 30 rain. After this period they were transferred to new vials with KRB for two sequential periods of 2 rain, then transferred to 40 mM K+-KRB for the first stimulation period of 2 rain, and then were again transferred to vials with KRB for 3 sequential periods of 2 rain. In some experiments, a second stimulation period was performed. To study Ca ++ dependence, Ca ++ was omitted from the medium and 0.1 mM EGTA was included during the washing period between the first and the second stimulation. Vials from basal and stimulus periods were removed from bath and kept frozen at - 20-°C until G A B A determination. At the end of the experiments both oviducts were homogenized as described for tissue samples to measure G A B A remainding in the tissue. In order to control the entire procedure, tissue slices obtained from the striatum were used. The striatum was freshly dissected and cut into 200 ~tm slices. Four slices were incubated in the same above conditions. To study the effect of temperature on G A B A efflux, oviducts were preincubated for 15 rain at 37-°C and then transferred to new vials for 60 rain incubation period at different temperature studied (4 -°, 20-0, 37'-'C). G A B A determinations: G A B A was analized by HPLC coupled with fluorometric detection. The HPLC unit was equiped with a mini pump (PM-11, LDC) to deliver the mobile phase. A Rheodyne injector (Model 7125) coupled to a 5 ~m reverse phase Biophase C18 column (250 x 4.5 ram, Bioanalytical Systems), and a fluorometric detector (Fluoromonitor III, LDC) with filters configured for an excitation wavelenght of 340-390 nm and an emission wavelenght of 490-700 nm. Tissue homogenates and superfusate samples were assayed for G A B A as previously described (19,20). For this purpose 100 ~tl of sample was mixed with
Vol.
52, No.
9, 1993
Oviductal
Fluid Volume
and G A B A E f f l u x
813
20 p.1 of 0.8 M borate buffer pH 10.5. Then the mixture was derivatized by adding 20 gl of orthophtaldehyde solution (OPA, Pierce) and 6-mercaptoethanol (Sigma) in methanol (20 mg OPA and 10 ul 13-mercaptoethanol in 5 ml methanol) and vortexed for 90 seconds. After the derivatization, 100 Ill of the mixture was injected through a 50 I.tl injection loop. The solvent used for the separation of GABA was 28 % acetonitrile, 0.1 M sodium phosphate, pH 5.5 adjusted with sodium hydroxide. After G A B A was eluted an 8 min washout step with 40% acetonitrile 60% water was performed.
Calculations and Statistical Analysis: Fractional release in each period was calculated and expressed as the percentage of total GABA present in the tissue. Total G A B A was calculated as the sum of the total GABA released during the incubation plus the amount remaining in the tissue after the experiment. The G A B A levels data were analyzed by one way analisys of variance followed by Duncan's post-hoc test for multiple comparison.
Results Subcellular fractionation: In oviducts subjected to a primary subcellular fractionation GABA was recovered as follows: the highest percentage (66.7 %) in the soluble fraction $3; almost 19.6 % in the microsomal fraction P3; 10.3 % in P2 and traces in P1 (table 1).
Endogenous G A B A efflux: Release of endogenous GABA from rat oviduct was studied by measuring G A B A efflux in different experimental conditions. In the presence of 2.5 mM K +, a spontaneous GABA efflux (about 0.5 % / m i n ) from the rat oviduct was observed. When the same tissue was subjected to depolarizing conditions, in the presence of 40 mM K ÷, there was not significant changes in the spontaneous GABA efflux. Other depolarizing conditions (12; 20 and 70 mM K ÷) were used, no one of which induced release of endogenous GABA (data not shown). When the depolarizing period was performed in Ca++-free medium plus 0.1 mM EGTA the spontaneous G A B A efflux remained unchanged ( Fig. 1A). 5mM Ba ~ was also used as a possible secretagoge but no effect was observed on G A B A efflux (data not shown). In order to control experimental conditions used striatal slices were subjected to the same depolarizing stimulus and a significant increase of GABA efflux was observed as shown in fig. lB. This increase in GABA efflux was completely dependent on the presence of Ca ++ in the incubation medium. Furthermore, spontaneous GABA effiux from the oviduct did not change with different incubation temperatures (Table 2). TABLE 1 Distribution of GABA and protein in primary subcellular fractions of rat oviduct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fractions
GABA (%)
Protein (%)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nuclear (P1) Mitocondrial (P2) Microsomal (P3) Soluble ($3)
2.23 10.34 19.62 66.69
+ 0.52 + 0.89 +_ 0.96 + 1.56
58.04 11.48 4.94 26.53
_+ 4.65 _+ 2.06 +_ 0.55 _+ 2.65
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Values are expressed as means _+ SEM of percentage of total activity of three independent experiments. G A B A levels: The GABA content in rat oviducts during postnatal development showed a progressive increase from day 5 reaching maximun values at days 35 to 40 (Fig. 2). The
814
Oviductal Fluid Volume and GABA Efflux
Vol.
52, No.
9, 1993
G A B A level at day one was undetectable with our procedure. Instead, the concentration of G A B A showed an increase up to day 10 followed by a decline at day 15, with a new increase until a maximun value at day 35 ( Fig 2). The G A B A levels in the rat oviduct fluctuated during the estrous cycle. A maximun value was obtained in proestrous while minimal values appeared in estrous phase. A decrease of 46% of G A B A concentration during the estrous in comparison to the proestrous phase was observed. Both G A B A content and G A B A concentration present the same pattern of changes (Fig 3).
40ramK*
40m~ K"
//
H
-
A x m
3
Q m
¢U
w
0
20-
0
0
m IO" L_
B 0
0
'
6~ (~
tim~in)e(m
Fig. 1 Comparison of G A B A efflux from estrous phase rat oviduct (A) and striatal slices (B). The spontaneous and K+-evoked endogenous G A B A efflux from tissue slices into the incubation medium, was measured in the same conditions during two consecutive incubation periods in the presence (continuous line) or absence of Ca ++(broken lines). Values in A represent the mean _+ SEM of three independent experiments. Values in B correspond to a single experiment. Discussion Several studies have provided evidences suggesting the existence of an extrinsic GABAergic inervation of the rat oviduct (6,7,14,15). In contrast, other studies have demonstrated that G A B A and GABAergic methabolic machinery are principally located in the epithelial cells of the mucosa, supporting a non-neuronal origin to oviductal G A B A (7,16,17). The data presented here show a spontaneous endogenuos G A B A efflux from the rat oviduct. This G A B A efflux was not affected by depolarization in the same conditions that
Vol.
52, No.
9,
1993
Oviductal
Fluid Volume
and G A B A E f f l u x
815
evoked a significant release of GABA from striatal slices. Moreover, Barium a well established secretagoge was also unable to induce any significant change in G A B A effiux from the rat oviduct and subcellular fractionation studies indicated that G A B A is principally located in the 130,000 xg supernatant. The results of the present study suggest that most of the oviductal G A B A is not present in a neuronal compartment. These results are in perfect agreement with previous studies of GABA localization (7,16). Furthermore, our results suggest that GABA is not located in a typical secretory system. In contrary to our results, Erdo et al (1986) have demonstrated that different depolarizing stimuli induce a significant increase in the 3H-GABA effiux from oviductal slices. However, their study was performed in rabbit oviductal slices using 3H-GABA that could label a different GABA compartment than the one studied in this paper. TABLE 2 Effect of incubation temperature on the spontaneous GABA efflux .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
temperature (-0C) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4 -0 20-0 37-0 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
GABA efflux (%) .
.
.
.
.
.
.
.
.
37.35 + 3.0 33.17 + 2.3 35.39 + 4.5 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
The data are expressed as percent of the total GABA present in the tissue. Values represent the mean + SEM of three independent experiments. In addition, the spontaneous GABA efflux seems a continuous passage of GABA from the tissue into the incubation medium rather than a carrier mediated system. This GABA effiux persists without significant changes during long periods of time (80 rain). The fractional outflow of GABA was the same in the two estrous cycle phases studied (estrous and diestrous phase). This G A B A efflux is not dependent on the incubation temperature. Furthermore, it has been demonstrated that rat oviducts show a very low capacity to take up 3H-GABA from the media (22). The experimental conditions used by Erdo and coworkers were such that should have favoured 3H-GABA influx if a carrier mediated system was operating (23, 24). All the evidences mentioned above strongly suggest that GABA transport from the epithelial cells of the rat oviduct is mediated by a pore rather than a carrier. Instead, in the case of rabbit oviduct, Erdo and Amenta (1986) have domonstrated the presence of a specific high-affinity GABA uptake system. Oviductal G A B A levels were examined during postnatal development of the rat. These G A B A levels showed a gradual increase reaching the highest values during prepuberal period. Prepuberal animals (35 days of age) in which ovarian steroid hormone levels are still low, showed a G A B A concentration very similar to that found in diestrous phase of adult rats. In agreement, Celotti et al. (1987) has reported that prepuberal rat oviducts present not only a G A B A content, but also a GAD activity analogous to those of diestrous rats. Interestingly, the same authors showed that in prepuberal animals, the GAD activity could be increased by the administration of gonadotropins or by high doses of ovarian steroids, whithout any change in the GABA content, suggesting that the enzymatic activity and the GABA concentration may be modulated by different mechanisms. The G A B A concentration showed an initial peak at 10 days, that may be produced by physiological related-changes during this period of development. Instead, G A B A content showed a steady continuous increase. A similar profile during development has been reported by Erdo (1986). Thus, the oviductal GABA content presents age related-changes in which ovarian steroid hormones appear not to be directly involved.
816
Oviductal
Fluid Volume
I
I
and G A B A E f f l u x
I
I
Vol.
I
,~
7(1
/
.3o
""J %\ \ :
!
ti
U
,n
,<
10
a.o ! 1
/
J7
'\a.-"'"
I'
/
/ / 10
9, 1993
F
/-/
U
m
I
0
52, No.
20
I
I
I
30
I
40
AGE (days)
Fig. 2 GABA levels in the rat oviduct during postnatal development. Values are the mean _+ SEM of 3-8 independent experiments. * significantly different from all other groups. ** significantly different whith value at 15 days. GABA level fluctuations during the estrous cycle has been previously reported, but results are contradictory. Martin del Rio (1981) found maximun GABA levels during diestrous phase. Meanwhile, other reports (9, 10, 11) have shown that the adult rat oviduct presents a maximun GABA content during proestrous phase. In the present study, GABA levels showed a significant fluctuation during the estrous cycle following a pattern very similar to that previously described in the rat oviduct (7,9,10). A significant decrease of GABA concentration in estrous, in comparison to proestrous phase was observed. These changes in oviductal GABA levels are consistent with the presence of high GABA content in ovarian bursa fluid during diestrous 1 phase (9). It is tempting to suggest that the oviductal GABA efflux is regulated by endocrine variables. Interestingly, the peak in GABA levels, during proestrous phase is coincident with the presence of high estrogen circulating levels. Even though the results of the present study and others suggest that estrogens are not directly involved in the control of the GABA efflux, it may be possible that this hormone indirectly enhances the efflux of GABA from the oviducts into the oviductal fluid, by an increase in the daily oviductal fluid production. This is supported by evidence that high circulating concentration of estrogens
Vol.
52, NO.
9, 1993
Oviductal
Fluid Volume
and G A B A E f f l u x
817
result in maximal production of oviductal fluid (for review see 25, 26, 27). Thus, the changes in oviductal fluid volume that physiologically occur during the estrous cycle could be modulating the amount of GABA present in this fluid.
100
I
10.0 !
80
8.0
"-
o
60
\
,," ".
4o
i
\.
",,,,,,,£ 4,0
20
2.0 m
diestrous 1 dlestrous 2 proestrous estrous ESTROUS CYCLE
Fig. 3 G A B A levels in the rat oviduct during the estrous cycle. Values are the mean _+ SEM of 3-6 independent experiments. * significantly different from all other group This neuronal passively neuronal neuronal
study has provided new evidence that GABA in the rat oviduct is mainly nonin nature. In fact, GABA is present in a non-releasable pool from where it can diffuse out to the oviductal fluid. Our results, do not rule out the possibility of a G A B A compartment that might be masked by the considerable size of the nonmucosal pool.
Finally, we propose the following working model to explain the results of this study and others in the literature: estrogen levels rising during proestrous phase induces a concomitant increase in the oviductal fluid volume. This increased fluid volume in turn will favour the diffusion of G A B A from the oviductal mucosa to the lumen. An increase diffusion of oviductal G A B A between proestrous and estrous phase could explain the observed decrease in oviductal G A B A content and the reported (7) enhance GAD activity in the estrous phase.
818
Oviductal
Fluid Volume
and G A B A E f f l u x
Vol.
52, No.
9, 1993
Further research is needed to asses a physiological role of GABA in the oviductal fluid. Possible functional roles to be considered are: ovum transport by the modulation of kinetocilia movements or a paracrine action upon the ovaries or uterus.
Acknowledgements The authors want to thank Ms Lucy Chacoff for helping during the preparation of the manuscript.
References 1. S.L. ERDO and J.R. WOLFF, J. Neurochem 54 336-372 (1990). 2. C. TANAKA and K. TANIYAMA, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 57-75,Raven Press, New York (1986). 3. S.L. ERDO and B.KISS, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds),5-17, Raven Press, New York (1986). 4. R. MARTIN DEL RIO, J. Biol.Chem. 256 9816-9819 (1981). 5. S.L. ERDO, B. ROSDY and L. SZPORNY, J. Neurochem. 38 1174-1176 (1982). 6. J.A. APUD, M.L. TAPPAZ, F. CELOTTI, P. NEGRI-CESI, C. MASOTTO and G. RACAGNI, J. Neurochem. 4__33121-125 (1984). 7. Y.L. MURASHIMA and T. KATO, J. Neurochem. 46 166-172 (1986). 8. N.J.K. TILLAKARATNE, M.G. ERLANDER, K.F. GRIEF, G.D. FRANTZ and A.J. TOBIN, Soc. Neurosci. Abstr. 16 695 (1990). 9. P. LOUZAN, M.G.P. GALLARDO and J.H. TRAMEZZANI, GABA and Endocrine Function, G. Racagni and A.O. Donoso (eds), 283-290, Raven Press, New York (1986). 10. S.L. ERDO, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 205- 221, Raven Press, New York (1986). 11. M.F. GIMENO, J. FERNANDEZ-PARDAL, M. VIGGIANO, M.C. PEZZOT and A.L. GIMENO, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 275-282, Raven Press, New York (1986). 12. S.L. ERDO, Life Sci. 3__441879-1884 (1984). 13. F. CELOTI'I, J.A. APUD, A.C. ROVESCALLI, P. NEGR1-CESI and G. RACAGNI, Endocrinology 120 700-706 (1987). 14. F. CELOTFI, J.A. APUD, R.C. MELCAGNI, C. MASOTTO, M. TAPPAZ and G. RACAGNI, Endocrinology 11___88334-339 (1986). 15. I. FERNANDEZ, R. MARTIN DEL RIO and L.M. OREZANZ, Life Sci. 3__fi61733-1737 (1985). 16. S.L. ERDO, J. SOMOGYI, J. HAMORI and F. AMENTA, Cell Tissue Res. 24,~ 621-626 (1986). 17. S.L. ERDO, F. JOO and J.R. WOLFF, Cell Tissue Res 255 431-434 (1989). 18. K. GYSLING and M.C. BEINFILD, Brain Research 407 110-116 (1987). 19. P. LINDROTH and K. MOPPER, Analytical Chemestry 51 1667-1674 (1979). 20. A. HAMBERGER, P. LINDROTH and B. NYSTROM, Brain Research 237 339-350 (1982). 21. S.L. Erdo and F. AMENTA, Eur. J. Pharmacol. 130 287-294 (1986). 22. S.L. ERDO, B. KISS, M.RIESZ and L. SZPORNY, Eur. J. Pharmacol. 130 295-303 (1986). 23. W.R. LIEB and W.D. STEIN, Biochim. Biophys. Acta 373 165-177 (1974). 24. W.R. LIEB and W.D. STEIN, Biochim. Biophys. Acta 373 178-196 (1974). 25. J.L. PERKINS, The Oviduct and its Functions, A.D. Johnson and C.W. Foley (eds), Academic Press, New York and London (1974). 26. B.G. BRACKETT and L. MASTROIANI JR, The Oviduct and its Functions, A.D. Johnson and C.W. Foley (eds), Academic Press, New York and London (1974). 27. G. OLIPHANT, The Fallopian Tube; Basic and Clinical Contribution, A.M. Siegler and M.D. Mountkisco, Futura Publishing Company Inc., New York (1986).
52, pp.
811-818
Pergamon
Press
NON-NEURONAL ENDOGENOUS GABA EFFLUX FROM THE RAT OVIDUCT Maria In~s Forray, Patricia Hidalgo, Felipe Diaz and Jorge Belmar Laboratory of Biochemical Pharmacology, Dept. Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Alameda 340, P.O. Box 114-D, Santiago, Chile. (Received
in final
form D e c e m b e r
15,
1992)
Summary The subcellular distribution of Gamma-aminobutyric acid (GABA) was studied in the rat oviduct. The highest content of GABA was found in the soluble fraction. The effect of chemical stimulation of the endogenous GABA effiux from the rat Qviduct was examined. High K + concentrations could not induce elevation of the GABA efflux. Instead, a continuous spontaneous GABA effiux without change for long periods of time was observed. The total GABA content and GABA concentration were determined in the rat oviduct on days 1, 5, 10, 15, 30, 35 and 40 of the postnatal period and also during the estrous cycle. During postnatal development the GABA levels increase gradually with time reaching at prepuberal age a concentration similar to that found in diestrous rats. In the estrous cycle both GABA content and GABA concentration reached the highest value in the proestrous and the lowest value in the estrous phase. These findings support the hypothesis that GABA effiux may be modulated by the changes in oviductal fluid volume during the estrous cycle. Gamma-aminobutyric acid (GABA) is considered in the central nervous system (CNS). The presence of in the peripheral organs and tissues motivate current plays a neurotransmitter role or whether some cells synthesize G A B A (for review see 1,2,3).
the principal inhibitory neurotransmitter this amino acid and its wide distribution interest in establishing whether GABA of these tissues and organs themselves
The rat oviduct contains high GABA amounts (4,5). Also, this organ contains significant glutamic acid descarboxylase (GAD) activity, the rate-limiting enzyme in the synthesis of G A B A in the CNS (6,7). This enzyme has two forms GAD 65 and GAD 67 encoded by two different genes. It has been shown that GAD 65 is also present in the rat oviduct (8). On the other hand, it has been shown that GABA levels or GAD activity change in different endocrine situations like estrous cycle (4,5,9,10,11), pregnancy (11,12), prepuberal period (13) and with different endocrine manipulations like ovariectomy and hypophysectomy (14,15). GABA secretion from the oviducts and its fluctuating level during estrous cycle in the ovarian bursa fluid were described by Louzan et al. (1986). The possibility that GABA could mediate the neuronal regulation of endocrine activity in the oviduct has also been studied. These studies have shown that G A B A content or GAD activity change after denervation procedures, such as vagotomy, subcutaneous transplantation of the oviduct (6) and section of the vascular nerve bundles (7,15). However, biochemical studies have shown the presence of highly concentrated GABA and GAD activity in oviductal mucosa and in the muscle layer (14), suggesting a non-neuronal nature for oviductal GABA. This is further supported by immunohystochemical evidences showing the presence of GABA, GABA-transaminase and GAD in the epithelial cells of the mucosa. GABA exhibit a specific localization in the basal Copyright
0 0 2 4 - 3 2 0 5 / 9 3 $6.00 + .00 © 1993 P e r g a m o n Press Ltd All r i g h t s
reserved.
812
O v i d u c t a l F l u i d V o l u m e and G A B A E f f l u x
Vol.
52, No.
9, 1993
body of the cilia (16,17). The present study was performed to elucidate the characteristics of the oviductal G A B A compartment and the possible mechanisms involved in G A B A secretion.
Materials and Methods Animals: Female Sprague-Dawley rats weighing 180-250 g were used, otherwise specified. For the postnatal level experiments, animals on day 1, 5, 10, 30, 35 and 40 of age were used weighing 10 to 110 g. The stages of the estrous cycle were assessed by vaginal smears examination and only animals presenting two or more consecutive 4 days cycles were used.
Tissue samples: The animals were sacrified, ovaries and oviducts were quickly removed, put at 4-0C and cleaned of connective tissue and fat. Oviducts were homogenized in ice cold 0.2 N perchloric acid with a glass-glass homogenizer and centrifugated for 20 rain at 12000 xg at 4-°C. The clear supernatant fractions were analized for G A B A content.
Subcellular fraetionation: Oviducts were homogenized in 10 % (w/v) buffered 0.32 M sucrose (pH 7.4) with a glass-glass homogenizer. This homogenate was subjected to differential centrifugation. The following fractions were obtained: a crude nuclear pellet (P1) by centrifugation at 800 xg for 10 rain.; a crude mitochondrial pellet (P2) by centrifugation at 8500 xg for 20 rain.; a microsomal pellet (P3) and a soluble fraction ($3) by centrifugation at 130,000 xg for 90 rain. All fractions were collected and kept frozen at - 20-°C until G A B A content was determined.
Endogenous G A B A efflux: For each experiment both oviducts obtained from rats on estrous or diestrous phase were cut in 8 pieces each and incubated in Krebs Ringer Bicarbonate solution (KRB) as previously described (18). For high K÷-KRB, equimolar amounts of NaC1 were replaced by KCI to maintain an iso-osmotic condition. Tissue were put in a stainless steel basket and preincubated in KRB for 30 rain. After this period they were transferred to new vials with KRB for two sequential periods of 2 rain, then transferred to 40 mM K+-KRB for the first stimulation period of 2 rain, and then were again transferred to vials with KRB for 3 sequential periods of 2 rain. In some experiments, a second stimulation period was performed. To study Ca ++ dependence, Ca ++ was omitted from the medium and 0.1 mM EGTA was included during the washing period between the first and the second stimulation. Vials from basal and stimulus periods were removed from bath and kept frozen at - 20-°C until G A B A determination. At the end of the experiments both oviducts were homogenized as described for tissue samples to measure G A B A remainding in the tissue. In order to control the entire procedure, tissue slices obtained from the striatum were used. The striatum was freshly dissected and cut into 200 ~tm slices. Four slices were incubated in the same above conditions. To study the effect of temperature on G A B A efflux, oviducts were preincubated for 15 rain at 37-°C and then transferred to new vials for 60 rain incubation period at different temperature studied (4 -°, 20-0, 37'-'C). G A B A determinations: G A B A was analized by HPLC coupled with fluorometric detection. The HPLC unit was equiped with a mini pump (PM-11, LDC) to deliver the mobile phase. A Rheodyne injector (Model 7125) coupled to a 5 ~m reverse phase Biophase C18 column (250 x 4.5 ram, Bioanalytical Systems), and a fluorometric detector (Fluoromonitor III, LDC) with filters configured for an excitation wavelenght of 340-390 nm and an emission wavelenght of 490-700 nm. Tissue homogenates and superfusate samples were assayed for G A B A as previously described (19,20). For this purpose 100 ~tl of sample was mixed with
Vol.
52, No.
9, 1993
Oviductal
Fluid Volume
and G A B A E f f l u x
813
20 p.1 of 0.8 M borate buffer pH 10.5. Then the mixture was derivatized by adding 20 gl of orthophtaldehyde solution (OPA, Pierce) and 6-mercaptoethanol (Sigma) in methanol (20 mg OPA and 10 ul 13-mercaptoethanol in 5 ml methanol) and vortexed for 90 seconds. After the derivatization, 100 Ill of the mixture was injected through a 50 I.tl injection loop. The solvent used for the separation of GABA was 28 % acetonitrile, 0.1 M sodium phosphate, pH 5.5 adjusted with sodium hydroxide. After G A B A was eluted an 8 min washout step with 40% acetonitrile 60% water was performed.
Calculations and Statistical Analysis: Fractional release in each period was calculated and expressed as the percentage of total GABA present in the tissue. Total G A B A was calculated as the sum of the total GABA released during the incubation plus the amount remaining in the tissue after the experiment. The G A B A levels data were analyzed by one way analisys of variance followed by Duncan's post-hoc test for multiple comparison.
Results Subcellular fractionation: In oviducts subjected to a primary subcellular fractionation GABA was recovered as follows: the highest percentage (66.7 %) in the soluble fraction $3; almost 19.6 % in the microsomal fraction P3; 10.3 % in P2 and traces in P1 (table 1).
Endogenous G A B A efflux: Release of endogenous GABA from rat oviduct was studied by measuring G A B A efflux in different experimental conditions. In the presence of 2.5 mM K +, a spontaneous GABA efflux (about 0.5 % / m i n ) from the rat oviduct was observed. When the same tissue was subjected to depolarizing conditions, in the presence of 40 mM K ÷, there was not significant changes in the spontaneous GABA efflux. Other depolarizing conditions (12; 20 and 70 mM K ÷) were used, no one of which induced release of endogenous GABA (data not shown). When the depolarizing period was performed in Ca++-free medium plus 0.1 mM EGTA the spontaneous G A B A efflux remained unchanged ( Fig. 1A). 5mM Ba ~ was also used as a possible secretagoge but no effect was observed on G A B A efflux (data not shown). In order to control experimental conditions used striatal slices were subjected to the same depolarizing stimulus and a significant increase of GABA efflux was observed as shown in fig. lB. This increase in GABA efflux was completely dependent on the presence of Ca ++ in the incubation medium. Furthermore, spontaneous GABA effiux from the oviduct did not change with different incubation temperatures (Table 2). TABLE 1 Distribution of GABA and protein in primary subcellular fractions of rat oviduct . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fractions
GABA (%)
Protein (%)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Nuclear (P1) Mitocondrial (P2) Microsomal (P3) Soluble ($3)
2.23 10.34 19.62 66.69
+ 0.52 + 0.89 +_ 0.96 + 1.56
58.04 11.48 4.94 26.53
_+ 4.65 _+ 2.06 +_ 0.55 _+ 2.65
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Values are expressed as means _+ SEM of percentage of total activity of three independent experiments. G A B A levels: The GABA content in rat oviducts during postnatal development showed a progressive increase from day 5 reaching maximun values at days 35 to 40 (Fig. 2). The
814
Oviductal Fluid Volume and GABA Efflux
Vol.
52, No.
9, 1993
G A B A level at day one was undetectable with our procedure. Instead, the concentration of G A B A showed an increase up to day 10 followed by a decline at day 15, with a new increase until a maximun value at day 35 ( Fig 2). The G A B A levels in the rat oviduct fluctuated during the estrous cycle. A maximun value was obtained in proestrous while minimal values appeared in estrous phase. A decrease of 46% of G A B A concentration during the estrous in comparison to the proestrous phase was observed. Both G A B A content and G A B A concentration present the same pattern of changes (Fig 3).
40ramK*
40m~ K"
//
H
-
A x m
3
Q m
¢U
w
0
20-
0
0
m IO" L_
B 0
0
'
6~ (~
tim~in)e(m
Fig. 1 Comparison of G A B A efflux from estrous phase rat oviduct (A) and striatal slices (B). The spontaneous and K+-evoked endogenous G A B A efflux from tissue slices into the incubation medium, was measured in the same conditions during two consecutive incubation periods in the presence (continuous line) or absence of Ca ++(broken lines). Values in A represent the mean _+ SEM of three independent experiments. Values in B correspond to a single experiment. Discussion Several studies have provided evidences suggesting the existence of an extrinsic GABAergic inervation of the rat oviduct (6,7,14,15). In contrast, other studies have demonstrated that G A B A and GABAergic methabolic machinery are principally located in the epithelial cells of the mucosa, supporting a non-neuronal origin to oviductal G A B A (7,16,17). The data presented here show a spontaneous endogenuos G A B A efflux from the rat oviduct. This G A B A efflux was not affected by depolarization in the same conditions that
Vol.
52, No.
9,
1993
Oviductal
Fluid Volume
and G A B A E f f l u x
815
evoked a significant release of GABA from striatal slices. Moreover, Barium a well established secretagoge was also unable to induce any significant change in G A B A effiux from the rat oviduct and subcellular fractionation studies indicated that G A B A is principally located in the 130,000 xg supernatant. The results of the present study suggest that most of the oviductal G A B A is not present in a neuronal compartment. These results are in perfect agreement with previous studies of GABA localization (7,16). Furthermore, our results suggest that GABA is not located in a typical secretory system. In contrary to our results, Erdo et al (1986) have demonstrated that different depolarizing stimuli induce a significant increase in the 3H-GABA effiux from oviductal slices. However, their study was performed in rabbit oviductal slices using 3H-GABA that could label a different GABA compartment than the one studied in this paper. TABLE 2 Effect of incubation temperature on the spontaneous GABA efflux .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
temperature (-0C) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4 -0 20-0 37-0 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
GABA efflux (%) .
.
.
.
.
.
.
.
.
37.35 + 3.0 33.17 + 2.3 35.39 + 4.5 .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
The data are expressed as percent of the total GABA present in the tissue. Values represent the mean + SEM of three independent experiments. In addition, the spontaneous GABA efflux seems a continuous passage of GABA from the tissue into the incubation medium rather than a carrier mediated system. This GABA effiux persists without significant changes during long periods of time (80 rain). The fractional outflow of GABA was the same in the two estrous cycle phases studied (estrous and diestrous phase). This G A B A efflux is not dependent on the incubation temperature. Furthermore, it has been demonstrated that rat oviducts show a very low capacity to take up 3H-GABA from the media (22). The experimental conditions used by Erdo and coworkers were such that should have favoured 3H-GABA influx if a carrier mediated system was operating (23, 24). All the evidences mentioned above strongly suggest that GABA transport from the epithelial cells of the rat oviduct is mediated by a pore rather than a carrier. Instead, in the case of rabbit oviduct, Erdo and Amenta (1986) have domonstrated the presence of a specific high-affinity GABA uptake system. Oviductal G A B A levels were examined during postnatal development of the rat. These G A B A levels showed a gradual increase reaching the highest values during prepuberal period. Prepuberal animals (35 days of age) in which ovarian steroid hormone levels are still low, showed a G A B A concentration very similar to that found in diestrous phase of adult rats. In agreement, Celotti et al. (1987) has reported that prepuberal rat oviducts present not only a G A B A content, but also a GAD activity analogous to those of diestrous rats. Interestingly, the same authors showed that in prepuberal animals, the GAD activity could be increased by the administration of gonadotropins or by high doses of ovarian steroids, whithout any change in the GABA content, suggesting that the enzymatic activity and the GABA concentration may be modulated by different mechanisms. The G A B A concentration showed an initial peak at 10 days, that may be produced by physiological related-changes during this period of development. Instead, G A B A content showed a steady continuous increase. A similar profile during development has been reported by Erdo (1986). Thus, the oviductal GABA content presents age related-changes in which ovarian steroid hormones appear not to be directly involved.
816
Oviductal
Fluid Volume
I
I
and G A B A E f f l u x
I
I
Vol.
I
,~
7(1
/
.3o
""J %\ \ :
!
ti
U
,n
,<
10
a.o ! 1
/
J7
'\a.-"'"
I'
/
/ / 10
9, 1993
F
/-/
U
m
I
0
52, No.
20
I
I
I
30
I
40
AGE (days)
Fig. 2 GABA levels in the rat oviduct during postnatal development. Values are the mean _+ SEM of 3-8 independent experiments. * significantly different from all other groups. ** significantly different whith value at 15 days. GABA level fluctuations during the estrous cycle has been previously reported, but results are contradictory. Martin del Rio (1981) found maximun GABA levels during diestrous phase. Meanwhile, other reports (9, 10, 11) have shown that the adult rat oviduct presents a maximun GABA content during proestrous phase. In the present study, GABA levels showed a significant fluctuation during the estrous cycle following a pattern very similar to that previously described in the rat oviduct (7,9,10). A significant decrease of GABA concentration in estrous, in comparison to proestrous phase was observed. These changes in oviductal GABA levels are consistent with the presence of high GABA content in ovarian bursa fluid during diestrous 1 phase (9). It is tempting to suggest that the oviductal GABA efflux is regulated by endocrine variables. Interestingly, the peak in GABA levels, during proestrous phase is coincident with the presence of high estrogen circulating levels. Even though the results of the present study and others suggest that estrogens are not directly involved in the control of the GABA efflux, it may be possible that this hormone indirectly enhances the efflux of GABA from the oviducts into the oviductal fluid, by an increase in the daily oviductal fluid production. This is supported by evidence that high circulating concentration of estrogens
Vol.
52, NO.
9, 1993
Oviductal
Fluid Volume
and G A B A E f f l u x
817
result in maximal production of oviductal fluid (for review see 25, 26, 27). Thus, the changes in oviductal fluid volume that physiologically occur during the estrous cycle could be modulating the amount of GABA present in this fluid.
100
I
10.0 !
80
8.0
"-
o
60
\
,," ".
4o
i
\.
",,,,,,,£ 4,0
20
2.0 m
diestrous 1 dlestrous 2 proestrous estrous ESTROUS CYCLE
Fig. 3 G A B A levels in the rat oviduct during the estrous cycle. Values are the mean _+ SEM of 3-6 independent experiments. * significantly different from all other group This neuronal passively neuronal neuronal
study has provided new evidence that GABA in the rat oviduct is mainly nonin nature. In fact, GABA is present in a non-releasable pool from where it can diffuse out to the oviductal fluid. Our results, do not rule out the possibility of a G A B A compartment that might be masked by the considerable size of the nonmucosal pool.
Finally, we propose the following working model to explain the results of this study and others in the literature: estrogen levels rising during proestrous phase induces a concomitant increase in the oviductal fluid volume. This increased fluid volume in turn will favour the diffusion of G A B A from the oviductal mucosa to the lumen. An increase diffusion of oviductal G A B A between proestrous and estrous phase could explain the observed decrease in oviductal G A B A content and the reported (7) enhance GAD activity in the estrous phase.
818
Oviductal
Fluid Volume
and G A B A E f f l u x
Vol.
52, No.
9, 1993
Further research is needed to asses a physiological role of GABA in the oviductal fluid. Possible functional roles to be considered are: ovum transport by the modulation of kinetocilia movements or a paracrine action upon the ovaries or uterus.
Acknowledgements The authors want to thank Ms Lucy Chacoff for helping during the preparation of the manuscript.
References 1. S.L. ERDO and J.R. WOLFF, J. Neurochem 54 336-372 (1990). 2. C. TANAKA and K. TANIYAMA, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 57-75,Raven Press, New York (1986). 3. S.L. ERDO and B.KISS, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds),5-17, Raven Press, New York (1986). 4. R. MARTIN DEL RIO, J. Biol.Chem. 256 9816-9819 (1981). 5. S.L. ERDO, B. ROSDY and L. SZPORNY, J. Neurochem. 38 1174-1176 (1982). 6. J.A. APUD, M.L. TAPPAZ, F. CELOTTI, P. NEGRI-CESI, C. MASOTTO and G. RACAGNI, J. Neurochem. 4__33121-125 (1984). 7. Y.L. MURASHIMA and T. KATO, J. Neurochem. 46 166-172 (1986). 8. N.J.K. TILLAKARATNE, M.G. ERLANDER, K.F. GRIEF, G.D. FRANTZ and A.J. TOBIN, Soc. Neurosci. Abstr. 16 695 (1990). 9. P. LOUZAN, M.G.P. GALLARDO and J.H. TRAMEZZANI, GABA and Endocrine Function, G. Racagni and A.O. Donoso (eds), 283-290, Raven Press, New York (1986). 10. S.L. ERDO, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 205- 221, Raven Press, New York (1986). 11. M.F. GIMENO, J. FERNANDEZ-PARDAL, M. VIGGIANO, M.C. PEZZOT and A.L. GIMENO, GABAergic Mechanisms in the Mammalian Periphery, S.L. Erdo and N.G. Bowery (eds), 275-282, Raven Press, New York (1986). 12. S.L. ERDO, Life Sci. 3__441879-1884 (1984). 13. F. CELOTI'I, J.A. APUD, A.C. ROVESCALLI, P. NEGR1-CESI and G. RACAGNI, Endocrinology 120 700-706 (1987). 14. F. CELOTFI, J.A. APUD, R.C. MELCAGNI, C. MASOTTO, M. TAPPAZ and G. RACAGNI, Endocrinology 11___88334-339 (1986). 15. I. FERNANDEZ, R. MARTIN DEL RIO and L.M. OREZANZ, Life Sci. 3__fi61733-1737 (1985). 16. S.L. ERDO, J. SOMOGYI, J. HAMORI and F. AMENTA, Cell Tissue Res. 24,~ 621-626 (1986). 17. S.L. ERDO, F. JOO and J.R. WOLFF, Cell Tissue Res 255 431-434 (1989). 18. K. GYSLING and M.C. BEINFILD, Brain Research 407 110-116 (1987). 19. P. LINDROTH and K. MOPPER, Analytical Chemestry 51 1667-1674 (1979). 20. A. HAMBERGER, P. LINDROTH and B. NYSTROM, Brain Research 237 339-350 (1982). 21. S.L. Erdo and F. AMENTA, Eur. J. Pharmacol. 130 287-294 (1986). 22. S.L. ERDO, B. KISS, M.RIESZ and L. SZPORNY, Eur. J. Pharmacol. 130 295-303 (1986). 23. W.R. LIEB and W.D. STEIN, Biochim. Biophys. Acta 373 165-177 (1974). 24. W.R. LIEB and W.D. STEIN, Biochim. Biophys. Acta 373 178-196 (1974). 25. J.L. PERKINS, The Oviduct and its Functions, A.D. Johnson and C.W. Foley (eds), Academic Press, New York and London (1974). 26. B.G. BRACKETT and L. MASTROIANI JR, The Oviduct and its Functions, A.D. Johnson and C.W. Foley (eds), Academic Press, New York and London (1974). 27. G. OLIPHANT, The Fallopian Tube; Basic and Clinical Contribution, A.M. Siegler and M.D. Mountkisco, Futura Publishing Company Inc., New York (1986).