- Email: [email protected]
Down-regulation of endogenous nitric oxide synthase in late-pregnancy and parturition in the rat hypothalamic magnocellular neurons and neurohypophysis
ELSEVIER
Neuroscience Letters 220 (1996) 133-136
Down-regulation of endogenous nitric oxide synthase in late-pregnancy and parturition in the rat hypothalamic magnocellular neurons and neurohypophysis Chuma
O. O k e r e * , T a k a s h i H i g u c h i
First Department of Physiology, Kochi Medical School. Nankoku. Kochi 783, Japan
Received 18 October 1996; revised version received 5 November 1996; accepted 15 November 1996
Abstract
Several recent lines of evidence suggest that nitric oxide (NO) may be an endogenous inhibitory regulator of the neurosecretory mechanism in magnocellular neurons of the paraventricular and the supraoptic nuclei in the hypothalamus. The NO synthase (NOS) system in the hypothalamo-neurohypophysial axis is regulated in an activity-dependent manner. The present study examined NOS activity in the magnocellular neurons and neurohypophysis during pregnancy and parturition by using the nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and assay of the specific NOS enzyme activity, respectively. In the paraventricular and supraoptic nuclei, the density and number of NADPH-diaphorase-positive ceils decreased in late-pregnancy and parturition. The specific activity of NOS in the neurohypophysis also decreased in late-pregnancy through parturition, and increased shortly afterward. Together with the ability of a NO donor to significantly delay the progress of parturition when administered centrally in parturient rats, these observations suggest that this down-regulation of NOS activity in the hypothalamo-neurohypophysial axis in late-pregnancy and parturition may be of physiological importance in the onset and/or progress of parturition. Copyright © 1996 Elsevier Science Ireland Ltd. Keywords: Magnocellular neurons; Nicotinamide adenine dinucleotide phosphate-diaphorase:
Nitric oxide; Nitric oxide synthase;
Parturition; Pregnancy
Nitric oxide (NO) initially identified as endotheliumderived relaxing factor has, in recent years, been shown to be an important neurochemical mediator molecule produced by the endothelial cells, macrophages and a host of other tissues [11]. Because NO is highly labile, its distribution in various tissues has been made possible by the localization of its more stable synthesizing enzyme, nitric oxide synthase (NOS). In appropriately fixed tissues, /3nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase reduces nitroblue tetrazolium to insoluble dark-blue formazan [9]. This convenient and reliable histochemical reaction has become important in neurobiology because neuronal NADPH-diaphorase is a NOS [6], and in paraformaldehyde-fixed tissues, selectively distinguishes constitutive NOS-positive cells [12]. * Corresponding author. TeL/fax: +81 888 802307; e-mail: [email protected]
In the hypothalamus, NOS is expressed in many neurosecretory cells and nuclei responsible for the regulation of reproductive processes such as the ventromedial nuclei, the median eminence, the preoptic and the periventricular nuclei, the bed nucleus of the stria terminalis and the parvo- and the magnocellular neurons of the paraventricular (PVN) and the supraoptic nuclei (SON) [1,2,19]. The magnocellular cells which secrete oxytocin and vasopressin contain NOS-positive neurons whose axons project to the neurohypophysis [18]. These NOS-positive neurons in the hypothalamo-neurohypophysial axis are regulated in an activity-dependent manner [7,8,16]. This raises the possibility of involvement of NO as an intercellular messenger in some functions of the hypothalamic neurosecretory cells. Evidence has been provided recently that central injection of the NO donor (sodium nitroprusside) or precursor (L-arginine) inhibits the transfer of milk from mother to pups and disrupts the characteristic milk ejec-
0304-3940/96/$12.00 Copyrigh! © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13248-1
134
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
B
~
b
perature ( 2 4 _ 1°C) conditions with unlimited access to food and water were used. NADPH-diaphorase activity was visualized as previously described [14] with 30 rain to 2 h duration of incubation. In addition, the neurohypophysis was harvested on ice from 30 guillotine-decapitated non-pregnant, pregnant or parturient rats, frozen in liquid nitrogen and stored at -80°C until assayed for NOS specific activity. The neurohypophysis was homogenized in 100 #l ice-cold buffer containing 1 mM EDTA and 50 mM Tris (pH 7.4). The homogenate was centrifuged at 12000 rev./min for 30 s at 4°C; 50 #l of the supernatant was incubated at 37°C for 6 min with 100/zl of reaction mixture containing 50 mM HEPES, 1 mM EDTA, 1.25 mM CaC12, 1 mM L-arginine, 10 mM flNADPH, 1 mM dithiothreitol, 1 mM tetrahydrobiopterin (Research Biochemicals, Inc.) and 1/zl of 1:4 dilution of 1 mCi/ml L-[2,3,4,5-aH]arginine hydrochloride (Amersham). The reaction was stopped with 900/~l buffer containing 100 mM HEPES (pH 5.5) and l0 mM EDTA (pH 7.4). The samples were respectively transferred to 1 ml columns of Dowex AG 50W-X8 resin (Na 2÷ form) equili-
Fig. 1. NADPH-diaphorase histochemistry in the paraventricular nucleus of non-pregnant (A), mid- pregnant (B), late-pregnant (C), parturient (D), 24 h postpartum (E) and lactating (F) rats. Note the decrease in NADPH-diaphorase-positive cluster of activity in the magnocellular subdivision (mpv) from late pregnancy through parturition, ppv, Parvocellular subdivision. Bar, 25/~m.
tion-associated burst of magnocellular (SON oxytocinergic) neurons. On the other hand, a NOS inhibitor significantly facilitated/permitted both responses [15]. In addition, central administration of sodium nitroprusside in parturient rats significantly delayed the progress of parturition; an observation that was not reversed by a single dose of oxytocin [ 13]. These observations suggest an inhibitory role of endogenous NO on the oxytocin neurosecretory mechanism. The present study was thus undertaken to test the hypothesis that the endogenous NOS system within the magnocellular hypothalamo-neurohypophysial axis may be down-regulated prior to and during parturition when the magnocellular neurosecretory activity is enhanced. Some parts of the present results have been published elsewhere in abstract form [14]. For NADPH-diaphorase histochemistry, non-pregnant (n = 14), mid-pregnant (day 12-17, n = 4), late-pregnant (day 19-21, n = 3), parturient (n = 3) and postpartum (n = 5) rats (Standard Wistar Strain; Japan SLC, Hamamatsu) housed under controlled light (0700-1900 h), tem-
Fig. 2. NADPH-diaphorase histochemistry in the supraoptic nucleus of non-pregnant (A), mid- pregnant (B), late-pregnant (C), parturient (D), 24 h postpartum (E) and lactating (F) rats. NADPH-diaphorase activity decreased from late-pregnancy through parturition. Bar, 25/~m.
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
NOS activity (pmol/mg/min) ?,,00
100
Non
Mid
Preg
Preg
Late Preg
Part.
6h
1,4 h
PP
PP
Fig. 3. Specific activity of NOS enzyme in the neurohypophysis in nonpregnant (non preg), mid-pregnant (mid preg), late-pregnant (late preg), parturient (part), within 6 h postpartum (6 h PP) and within 24 h postpartum (24 h PP) rats. Data are presented as mean + SEM of three to seven animals. *P < 0.05 versus rton-pregnant rats (Mann-Whitney Utest).
brated with stop buffer and eluted with 1 ml ddH20. One millilitre of elute containing [3H]citrulline was added into a tube containing 5 ml scintillation liquid (Aquasol, Packard Chem.) and counted with a liquid scintillation analyzer (Packard 2500). The protein content of the homogenate was determined by the Lowry method using BCA protein assay kit (Pierce, IL, USA). Specific NOS activity was expressed as pmol/mg per rain [3H]citrulline. Data are presented as mean + SEM and analyzed statistically using the Mann-Whitney U-test. NADPH-diaphorase activity was detected in both the parvocellular and magnocellular subdivisions of the PVN (Fig. 1). In mid-pregnant rats, the density of NADPH-diaphorase-positive neurons in the magnocellular subdivision was similar to that in non-pregnant rats (Fig. 1A,B). However, in late-pregnancy and during parturition, a significant decrease in the density of NADPH-diaphorase-positive cells was observed (Fig. IC,D). Such decrease was also observed in the rats which were sacrificed within 30 min and 2 h postpartum (data not shown). In the rats sacrificed 24 h after parturition and during lactation, the density and number of NADPH-diaphorase-positive cells formed a dense meshwork similar to that observed in non-pregnant or mid-pregnant rats (Fig. 1E,F). Such changes in NADPH-diaphorase activity were observed in the SON (Fig. 2). During the normal estrous cycle, no apparent changes in NADPH-diaphorase activity were detected in both the PVN and the SON (data not shown). The specific activity of NOS was also examined in the neurohypophysis during pregnancy and parturition (Fig. 3). No significant change in specific NOS activity was
135
detected until in late pregnancy. This decrease in NOS activity persisted through parturition, but within 6 h after parturition, the activity was not significantly different from that in the non-pregnant or mid-pregnant rats. The results of the present study have indicated that the endogenous NOS system within the magnocellular hypothalamo-neurohypophysial system is down-regulated in late-pregnancy and parturition. The magnocellular neurons in the PVN and the SON synthesize oxytocin and vasopressin; oxytocin is an important mediator of reproductive events, particularly of parturition. During latepregnancy, oxytocin mRNA and peptide are increased in the PVN and the SON [4,17,20] with associated increase in neurohypophysial oxytocin stores [5]. Some recent evidence of function-dependent changes in NOS in the hypothalamo-neurohypophysial system suggest a neuromodulatory role of NO as a regulator of oxytocin and vasopressin secretion [7,16]. More than 95% of oxytocin-immunoreactive neurons in the PVN and the SON showed NADPH-diaphorase activity; whereas few, if any, vasopressin-immunoreactive neurons showed NADPHdiaphorase activity; an observation interpreted as suggesting that oxytocin neurons are the main source of NO production in the hypothalamic-pituitary pathway [10]. The release of vasopressin and oxytocin from the neurohypophysis may be influenced by their colocalized substances [3] and it has been suggested that NO synthesized in oxytocinergic neurons may participate in auto- and/or crossregulation of oxytocin and vasopressin release [10]. Changes of NOS activity in the magnocellular neurons and neurohypophysis in late-pregnancy observed in the present study might reflect the build-up of oxytocin stores in late-pregnancy and the release of oxytocin during parturition. The endogenous NOS system may exert an inhibitory influence upon the magnocellular (oxytocin) secretory mechanism. The decrease in NOS activity in late-pregnancy and parturition may release the magnocellular secretory system from this inhibition. In support of this assumption, intracerebroventricular or (chronic) systemic injection of a NO donor in parturient or late-pregnant rats respectively, significantly prolongs the duration of parturition [13]. C.O. Okere and this study were respectively supported by a scholarship and research grants (to T. Higuchi) from the Ministry of Education, Science, Culture and Sports of Japan. [1] Bhat+ G. and Mahesh, V., Lamar., Ping, L., Aguan, K. and Binrann, D.W., Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing homaone and co-localization with N-methyI-D-aspartate receptors, Neuroendocrinology, 62 (1995) 187-197. [2] Bhat, G., Mahesh, V.B., Aguan, K. and Brann, D.W., Evidence that brain nitric oxide is the major nitric oxide synthase in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels, Neuroendocrinology, 64 (1996) 9 3 102.
136
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
[3] BickneU, R.J., Chapman, C. and Leng, G., Effect of opioid agonists and antagonists on oxytocin and vasopressin release in vitro, Neuroendocrinology, 41 (1985) 142-148. [4] CaldweU, J.D., Greer, E.R., Johnson, M.F., Prange, A.J. and Pedersen, C.A., Oxytocin and vasopressin immunoreactivity in hypothalamic and extrahypothalamic sites in late pregnancy and postpartum rats, Neuroendocrinology, 46 (1987) 39-47. [5] Fuchs, A.R. and Saito, S., Pituitary oxytocin and vasopressin content of pregnant rats before, during and after parturition, Endocrinology, 88 (1971) 574-578. [6] Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. [7] Kadowaki, K., Kishimoto, J., Leng, G. and Emson, P.C., Up-regulation of nitric oxide synthase (NOS) gene-expression together with NOS activity in the rat hypothalamo-hypophyseal system after chronic salt loading - evidence of a neuromodulatory role of nitric oxide in arginine-vasopressin and oxytocin secretion, Endocrinology, 134 (1994) 1011-1017. [8] Kishimoto, J., Tsuchiya, T., Emson, P.C. and Nakayama, Y., Immobilization-induoed stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitaryadrenal axis in rats, Brain Res., 720 (1996) 159-171. [9] Kuonen, D.R., Kemp, M.C. and Roberts, P.J., Demonstration and biochemical characterization of rat brain NADPH-dependent diaphorase, J. Neurochem., 50 (1988) 1017-1025. [10] Miyagawa, A., Okamura, H. and Ibata, Y., Coexistence of oxytocin and NADPH-diaphorase in magnocellular neurons of the paraventricular and the supraoptic nuclei of the rat hypothalamus, Neurosci. Lett., 171 (1994) 13-16. [11] Moncada, S., Palmer, R.M.J. and Higgs, E.A., Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev., 43 (1991) 109-142.
[12] Nakos, G. and Gossrau, R., When NADPH diaphorase works in the presence of formaldehyde, the enzyme appears to selectively visualize cells with constitutive nitric oxide synthase, Acta Histochem., 96 (1994) 335-343. [13] Okere, C.O., Higuchi, T., Kaba, H., Russel, J.A., Okutani, F., Takahashi, S. and Murata, T., Nitric oxide prolongs parturition and inhibits maternal behavior in rats, NeuroReport, 7 (1996) 1695- 1699. [14] Okere, C.O., Takahashi, S., Murata, T., Okutani, F. and Higuchi, T., NADPH-diaphorase staining suggests variation of nitric oxide synthase during pregnancy and parturition in the rat anterior pituitary, paraventricular and supraoptic nuclei, Acta Histochem. Cytocbem., 29 Suppl. (1996) 1045-1046. [15] Okere, C.O., Wang, Y.-F., Higuchi, T., Negoro, H., Okutani, F., Takahashi, S. and Murata, T., The effect of systemic and central nitric oxide administration on milk availability in lactating rats, NeuroReport, (1996) in press. [16] Pow, D.V., NADPH-diaphorase (nitric oxide synthase) staining in the rat supraoptic nucleus is activity dependent: possible functional implications, J. Neuroendocrinol., 4 (1992) 377-380. [17] Van Tol, H.H.M.E., Bolwerk, E.L.M., Liu, B. and Burbach, J.P.H., Oxytocin and vasopressin gene expression in the hypothalamo-neurohypophysial system of the rat during the estrus cycle, pregnancy and lactation, Endocrinology, 122 (1988) 945-951. [18] Vanhatalo, S. and Soinila, S., Nitric oxide synthase in the hypothalamo-pituitary pathways, J. Chem. Neuroanat., 8 (1995) 165173. [19] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755784. [20] Zingg, H.H. and Lefebvre, D.L., Oxytocin and vasopressin gene expression during gestation and lactation, Mol. Brain Res., 4 (1988) 1-6.
Neuroscience Letters 220 (1996) 133-136
Down-regulation of endogenous nitric oxide synthase in late-pregnancy and parturition in the rat hypothalamic magnocellular neurons and neurohypophysis Chuma
O. O k e r e * , T a k a s h i H i g u c h i
First Department of Physiology, Kochi Medical School. Nankoku. Kochi 783, Japan
Received 18 October 1996; revised version received 5 November 1996; accepted 15 November 1996
Abstract
Several recent lines of evidence suggest that nitric oxide (NO) may be an endogenous inhibitory regulator of the neurosecretory mechanism in magnocellular neurons of the paraventricular and the supraoptic nuclei in the hypothalamus. The NO synthase (NOS) system in the hypothalamo-neurohypophysial axis is regulated in an activity-dependent manner. The present study examined NOS activity in the magnocellular neurons and neurohypophysis during pregnancy and parturition by using the nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry and assay of the specific NOS enzyme activity, respectively. In the paraventricular and supraoptic nuclei, the density and number of NADPH-diaphorase-positive ceils decreased in late-pregnancy and parturition. The specific activity of NOS in the neurohypophysis also decreased in late-pregnancy through parturition, and increased shortly afterward. Together with the ability of a NO donor to significantly delay the progress of parturition when administered centrally in parturient rats, these observations suggest that this down-regulation of NOS activity in the hypothalamo-neurohypophysial axis in late-pregnancy and parturition may be of physiological importance in the onset and/or progress of parturition. Copyright © 1996 Elsevier Science Ireland Ltd. Keywords: Magnocellular neurons; Nicotinamide adenine dinucleotide phosphate-diaphorase:
Nitric oxide; Nitric oxide synthase;
Parturition; Pregnancy
Nitric oxide (NO) initially identified as endotheliumderived relaxing factor has, in recent years, been shown to be an important neurochemical mediator molecule produced by the endothelial cells, macrophages and a host of other tissues [11]. Because NO is highly labile, its distribution in various tissues has been made possible by the localization of its more stable synthesizing enzyme, nitric oxide synthase (NOS). In appropriately fixed tissues, /3nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase reduces nitroblue tetrazolium to insoluble dark-blue formazan [9]. This convenient and reliable histochemical reaction has become important in neurobiology because neuronal NADPH-diaphorase is a NOS [6], and in paraformaldehyde-fixed tissues, selectively distinguishes constitutive NOS-positive cells [12]. * Corresponding author. TeL/fax: +81 888 802307; e-mail: [email protected]
In the hypothalamus, NOS is expressed in many neurosecretory cells and nuclei responsible for the regulation of reproductive processes such as the ventromedial nuclei, the median eminence, the preoptic and the periventricular nuclei, the bed nucleus of the stria terminalis and the parvo- and the magnocellular neurons of the paraventricular (PVN) and the supraoptic nuclei (SON) [1,2,19]. The magnocellular cells which secrete oxytocin and vasopressin contain NOS-positive neurons whose axons project to the neurohypophysis [18]. These NOS-positive neurons in the hypothalamo-neurohypophysial axis are regulated in an activity-dependent manner [7,8,16]. This raises the possibility of involvement of NO as an intercellular messenger in some functions of the hypothalamic neurosecretory cells. Evidence has been provided recently that central injection of the NO donor (sodium nitroprusside) or precursor (L-arginine) inhibits the transfer of milk from mother to pups and disrupts the characteristic milk ejec-
0304-3940/96/$12.00 Copyrigh! © 1996 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(96)13248-1
134
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
B
~
b
perature ( 2 4 _ 1°C) conditions with unlimited access to food and water were used. NADPH-diaphorase activity was visualized as previously described [14] with 30 rain to 2 h duration of incubation. In addition, the neurohypophysis was harvested on ice from 30 guillotine-decapitated non-pregnant, pregnant or parturient rats, frozen in liquid nitrogen and stored at -80°C until assayed for NOS specific activity. The neurohypophysis was homogenized in 100 #l ice-cold buffer containing 1 mM EDTA and 50 mM Tris (pH 7.4). The homogenate was centrifuged at 12000 rev./min for 30 s at 4°C; 50 #l of the supernatant was incubated at 37°C for 6 min with 100/zl of reaction mixture containing 50 mM HEPES, 1 mM EDTA, 1.25 mM CaC12, 1 mM L-arginine, 10 mM flNADPH, 1 mM dithiothreitol, 1 mM tetrahydrobiopterin (Research Biochemicals, Inc.) and 1/zl of 1:4 dilution of 1 mCi/ml L-[2,3,4,5-aH]arginine hydrochloride (Amersham). The reaction was stopped with 900/~l buffer containing 100 mM HEPES (pH 5.5) and l0 mM EDTA (pH 7.4). The samples were respectively transferred to 1 ml columns of Dowex AG 50W-X8 resin (Na 2÷ form) equili-
Fig. 1. NADPH-diaphorase histochemistry in the paraventricular nucleus of non-pregnant (A), mid- pregnant (B), late-pregnant (C), parturient (D), 24 h postpartum (E) and lactating (F) rats. Note the decrease in NADPH-diaphorase-positive cluster of activity in the magnocellular subdivision (mpv) from late pregnancy through parturition, ppv, Parvocellular subdivision. Bar, 25/~m.
tion-associated burst of magnocellular (SON oxytocinergic) neurons. On the other hand, a NOS inhibitor significantly facilitated/permitted both responses [15]. In addition, central administration of sodium nitroprusside in parturient rats significantly delayed the progress of parturition; an observation that was not reversed by a single dose of oxytocin [ 13]. These observations suggest an inhibitory role of endogenous NO on the oxytocin neurosecretory mechanism. The present study was thus undertaken to test the hypothesis that the endogenous NOS system within the magnocellular hypothalamo-neurohypophysial axis may be down-regulated prior to and during parturition when the magnocellular neurosecretory activity is enhanced. Some parts of the present results have been published elsewhere in abstract form [14]. For NADPH-diaphorase histochemistry, non-pregnant (n = 14), mid-pregnant (day 12-17, n = 4), late-pregnant (day 19-21, n = 3), parturient (n = 3) and postpartum (n = 5) rats (Standard Wistar Strain; Japan SLC, Hamamatsu) housed under controlled light (0700-1900 h), tem-
Fig. 2. NADPH-diaphorase histochemistry in the supraoptic nucleus of non-pregnant (A), mid- pregnant (B), late-pregnant (C), parturient (D), 24 h postpartum (E) and lactating (F) rats. NADPH-diaphorase activity decreased from late-pregnancy through parturition. Bar, 25/~m.
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
NOS activity (pmol/mg/min) ?,,00
100
Non
Mid
Preg
Preg
Late Preg
Part.
6h
1,4 h
PP
PP
Fig. 3. Specific activity of NOS enzyme in the neurohypophysis in nonpregnant (non preg), mid-pregnant (mid preg), late-pregnant (late preg), parturient (part), within 6 h postpartum (6 h PP) and within 24 h postpartum (24 h PP) rats. Data are presented as mean + SEM of three to seven animals. *P < 0.05 versus rton-pregnant rats (Mann-Whitney Utest).
brated with stop buffer and eluted with 1 ml ddH20. One millilitre of elute containing [3H]citrulline was added into a tube containing 5 ml scintillation liquid (Aquasol, Packard Chem.) and counted with a liquid scintillation analyzer (Packard 2500). The protein content of the homogenate was determined by the Lowry method using BCA protein assay kit (Pierce, IL, USA). Specific NOS activity was expressed as pmol/mg per rain [3H]citrulline. Data are presented as mean + SEM and analyzed statistically using the Mann-Whitney U-test. NADPH-diaphorase activity was detected in both the parvocellular and magnocellular subdivisions of the PVN (Fig. 1). In mid-pregnant rats, the density of NADPH-diaphorase-positive neurons in the magnocellular subdivision was similar to that in non-pregnant rats (Fig. 1A,B). However, in late-pregnancy and during parturition, a significant decrease in the density of NADPH-diaphorase-positive cells was observed (Fig. IC,D). Such decrease was also observed in the rats which were sacrificed within 30 min and 2 h postpartum (data not shown). In the rats sacrificed 24 h after parturition and during lactation, the density and number of NADPH-diaphorase-positive cells formed a dense meshwork similar to that observed in non-pregnant or mid-pregnant rats (Fig. 1E,F). Such changes in NADPH-diaphorase activity were observed in the SON (Fig. 2). During the normal estrous cycle, no apparent changes in NADPH-diaphorase activity were detected in both the PVN and the SON (data not shown). The specific activity of NOS was also examined in the neurohypophysis during pregnancy and parturition (Fig. 3). No significant change in specific NOS activity was
135
detected until in late pregnancy. This decrease in NOS activity persisted through parturition, but within 6 h after parturition, the activity was not significantly different from that in the non-pregnant or mid-pregnant rats. The results of the present study have indicated that the endogenous NOS system within the magnocellular hypothalamo-neurohypophysial system is down-regulated in late-pregnancy and parturition. The magnocellular neurons in the PVN and the SON synthesize oxytocin and vasopressin; oxytocin is an important mediator of reproductive events, particularly of parturition. During latepregnancy, oxytocin mRNA and peptide are increased in the PVN and the SON [4,17,20] with associated increase in neurohypophysial oxytocin stores [5]. Some recent evidence of function-dependent changes in NOS in the hypothalamo-neurohypophysial system suggest a neuromodulatory role of NO as a regulator of oxytocin and vasopressin secretion [7,16]. More than 95% of oxytocin-immunoreactive neurons in the PVN and the SON showed NADPH-diaphorase activity; whereas few, if any, vasopressin-immunoreactive neurons showed NADPHdiaphorase activity; an observation interpreted as suggesting that oxytocin neurons are the main source of NO production in the hypothalamic-pituitary pathway [10]. The release of vasopressin and oxytocin from the neurohypophysis may be influenced by their colocalized substances [3] and it has been suggested that NO synthesized in oxytocinergic neurons may participate in auto- and/or crossregulation of oxytocin and vasopressin release [10]. Changes of NOS activity in the magnocellular neurons and neurohypophysis in late-pregnancy observed in the present study might reflect the build-up of oxytocin stores in late-pregnancy and the release of oxytocin during parturition. The endogenous NOS system may exert an inhibitory influence upon the magnocellular (oxytocin) secretory mechanism. The decrease in NOS activity in late-pregnancy and parturition may release the magnocellular secretory system from this inhibition. In support of this assumption, intracerebroventricular or (chronic) systemic injection of a NO donor in parturient or late-pregnant rats respectively, significantly prolongs the duration of parturition [13]. C.O. Okere and this study were respectively supported by a scholarship and research grants (to T. Higuchi) from the Ministry of Education, Science, Culture and Sports of Japan. [1] Bhat+ G. and Mahesh, V., Lamar., Ping, L., Aguan, K. and Binrann, D.W., Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing homaone and co-localization with N-methyI-D-aspartate receptors, Neuroendocrinology, 62 (1995) 187-197. [2] Bhat, G., Mahesh, V.B., Aguan, K. and Brann, D.W., Evidence that brain nitric oxide is the major nitric oxide synthase in the hypothalamus of the adult female rat and that nitric oxide potently regulates hypothalamic cGMP levels, Neuroendocrinology, 64 (1996) 9 3 102.
136
C.O. Okere, T. Higuchi / Neuroscience Letters 220 (1996) 133-136
[3] BickneU, R.J., Chapman, C. and Leng, G., Effect of opioid agonists and antagonists on oxytocin and vasopressin release in vitro, Neuroendocrinology, 41 (1985) 142-148. [4] CaldweU, J.D., Greer, E.R., Johnson, M.F., Prange, A.J. and Pedersen, C.A., Oxytocin and vasopressin immunoreactivity in hypothalamic and extrahypothalamic sites in late pregnancy and postpartum rats, Neuroendocrinology, 46 (1987) 39-47. [5] Fuchs, A.R. and Saito, S., Pituitary oxytocin and vasopressin content of pregnant rats before, during and after parturition, Endocrinology, 88 (1971) 574-578. [6] Hope, B.T., Michael, G.J., Knigge, K.M. and Vincent, S.R., Neuronal NADPH-diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811-2814. [7] Kadowaki, K., Kishimoto, J., Leng, G. and Emson, P.C., Up-regulation of nitric oxide synthase (NOS) gene-expression together with NOS activity in the rat hypothalamo-hypophyseal system after chronic salt loading - evidence of a neuromodulatory role of nitric oxide in arginine-vasopressin and oxytocin secretion, Endocrinology, 134 (1994) 1011-1017. [8] Kishimoto, J., Tsuchiya, T., Emson, P.C. and Nakayama, Y., Immobilization-induoed stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamic-pituitaryadrenal axis in rats, Brain Res., 720 (1996) 159-171. [9] Kuonen, D.R., Kemp, M.C. and Roberts, P.J., Demonstration and biochemical characterization of rat brain NADPH-dependent diaphorase, J. Neurochem., 50 (1988) 1017-1025. [10] Miyagawa, A., Okamura, H. and Ibata, Y., Coexistence of oxytocin and NADPH-diaphorase in magnocellular neurons of the paraventricular and the supraoptic nuclei of the rat hypothalamus, Neurosci. Lett., 171 (1994) 13-16. [11] Moncada, S., Palmer, R.M.J. and Higgs, E.A., Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev., 43 (1991) 109-142.
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