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Endocrinology Vol. 140, No. 1 286-291
Copyright © 1999 by The Endocrine Society

ARTICLES

Nitric Oxide Plays an Important Role in the Diurnal Change of Tuberoinfundibular Dopaminergic Neuronal Activity and Prolactin Secretion in Ovariectomized, Estrogen/Progesterone-Treated Rats1

Shih-Hui YEN and Jenn-Tser Pan

Department of Physiology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 11221

Address all correspondence and requests for reprints to: Jenn-Tser Pan, Ph.D., Department of Physiology, National Yang-Ming University, Taipei, Taiwan 11221.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A significant diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity coincident with the estrogen (E2)-induced afternoon PRL surge has been reported in ovariectomized, E2-primed (OVX+E2) rats. Systemic injection of a nitric oxide (NO) synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NA, 50 mg/kg, ip at 1000 and 1200 h), significantly blocked the diurnal changes of TIDA neuronal activity and PRL secretion at 1500 and 1700 h in OVX+E2 rats. Coadministration of L-arginine (300 mg/kg, ip) with L-NA completely prevented the effects of L-NA. Total nitrite/nitrate levels in the serum of L-NA- and L-NA+L-arginine-treated rats substantiated the effects of L-NA and L-arginine on NO production. Pretreatment of antisense oligodeoxynucleotide (ODN; 1 µg/3 µl; intracerebroventricularly at 48, 24, and 7 h before sacrifice) against the messenger RNA (mRNA) of constitutive NOS, i.e. neuronal NOS or endothelial NOS, was also effective in preventing the diurnal changes of TIDA neuronal activity and PRL surge at 1500 h. The same treatment of antisense ODN against the mRNA of inducible NOS, i.e. macrophage NOS, had no effect.

Progesterone (P4) has been reported to advance and augment the diurnal changes of TIDA neuronal activity and the afternoon PRL surge, by 1 h, in both proestrous and OVX+E2 rats. We further showed that L-NA dose dependently (50 but not 5 mg/kg, ip at 1000 and 1200 h) blocked the effect of P4 on TIDA neurons and serum PRL at 1300 h, which effect could be negated by simultaneous administration of L-arginine (300 mg/kg, ip). Pretreatment with antisense ODNs against the mRNA of neuronal NOS or endothelial NOS, but not macrophage NOS, was also effective in preventing the P4’s effect on TIDA neuronal activity and PRL secretion at 1300 h. In summary, NO may play a physiological role in the E2- and P4-regulated diurnal changes of TIDA neuronal activity and PRL secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO), a newly identified gaseous neurotransmitter, has been shown to play a significant role in the ovarian steroid-induced surges of LH and PRL in female rats (1, 2, 3). Increasing the NO production by pharmacological means, i.e. using NO donors, can stimulate PRL secretion in conscious male rats (4); whereas modification of endogenous NO by L-arginine or N{omega}-nitro-L-arginine-methyl ester does not have a significant effect (4). In addition, NO may also be involved in stress- and morphine-induced PRL secretion (5).

The mechanism for NO-stimulated LH release has been shown to involve increased release of LHRH (1, 6). The one for NO-induced PRL secretion, however, has not been reported. Unlike that of LH, the secretion of PRL is predominantly inhibited by dopamine (DA) released from the tuberoinfundibular dopaminergic (TIDA) neurons (7). This laboratory (8, 9) has previously reported that a diurnal change of TIDA neuronal activity exists in female rats, which is essential for the estrogen (E2)-induced afternoon PRL surge. We (10) further found that progesterone (P4) can advance and augment this diurnal change of TIDA neuronal activity and PRL secretion in ovariectomized, E2-primed (OVX + E2) rats and in proestrus rats. Whether NO is involved in the diurnal change of TIDA neuronal activity and PRL secretion in E2-primed female rats and in the action of P4 were the primary aims of this study.

To answer these questions, we used a potent NO synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NA) in this study. Because there are three major types of NOS that have been identified to date [i.e. neuronal NOS (nNOS), endothelial NOS (eNOS), and macrophage NOS (mNOS) (11, 12)], we then used antisense oligodeoxynucleotides (ODN) against each of them in this study. The TIDA neuronal activity was determined by measuring the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), the major metabolite of DA, in the median eminence (ME); and serum PRL was determined by RIA. The use of the ME DOPAC level as an index for TIDA neuronal activity has been validated in previous studies (7, 8, 9, 10, 13, 14). Though it is not the most sensitive index, its correlation with the concurrent serum PRL level in the same animal makes it the method of choice in this study. Total nitrite/nitrate levels in the sera of L-NA- and L-NA+L-arginine-treated rats were also determined, to assess the effects of L-NA and L-arginine on the NO synthesis in the body (15, 16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Adult female Sprague Dawley rats, weighing 220–250 g, were purchased from the University Animal Center and housed in a temperature (23 ± 1 C)- and light (lights on from 0600 to 2000 h)-controlled room with free access to rat chow and tap water. All rats were subjected to surgical ovariectomy and, 1 week later, implanted with sc capsules (silicone tubing, A-M Systems, Everett, WA; id, 1.57 mm; od, 3.18 mm; active length, 20 mm) containing 17ß-estradiol (E2; Sigma Chemical Co., St. Louis, MO; 150 mg/ml corn oil; Sigma Chemical Co.) for 6 more days before they were used for experimentation. P4 (Sigma Chemical Co.; 2 mg/kg, dissolved in corn oil containing 2% ethanol) was given sc to certain groups of rats at 0800 h on the experiment day. The dosages of E2 and P4 used were adopted from previous studies (10, 17).

In the studies, L-NA (RBI, Natick, MA; 5 or 50 mg/kg) and L-arginine (Sigma Chemical Co.; 300 mg/kg, the precursor of NO) were injected ip at specific times on the day of the experiment. L-arginine was dissolved and kept in warmed saline (37 C) during injection. In the study that used intracerebroventricular (icv) injection of ODNs, each rat was implanted with a cannula (23-gauge stainless steel tubing, 10 mm in length) in its right lateral cerebroventricle, 6 days before the experiment (the same time that the E2-capsule was implanted). Ether and equithesin were used as anesthetics for ovariectomy and stereotaxic surgery, respectively.

All rats were killed by decapitation, at specific time points during the day, with no anesthesia. Extra care was taken to minimize stress to the animal: the surgery room is connected with the animal room with a door, and the time between picking up the rat in one room and decapitation in another was less than 10 sec. After decapitation, trunk blood was collected and chilled on ice; the brain was quickly removed from its skull and frozen on dry ice. Thick (600 µm), coronal brain sections were prepared with a tabletop microtome and thaw-mounted onto glass slides. The ME region was removed from the sections, using a modified micropunch technique (13), and was stored in 40 µl of 0.15 M sodium phosphate buffer containing 0.65 mM sodium octanesulfonate, 0.5 mM EDTA, 12% ethanol, at pH 2.6. Both ME and serum samples were stored at -20 C until assayed.

Experimental design
In the first experiment, the OVX+E2 rats were divided into four groups and were decapitated at 1000, 1300, 1500, or 1700 h. Except for the group killed at 1000 h, half of the other groups were treated with L-NA (50 mg/kg, ip) twice, at 1000 and 1200 h, and were decapitated at 1300, 1500, or 1700 h, along with their vehicle-treated controls.

In the second experiment, the OVX+E2 rats were divided into four groups, and each received injections of the vehicle, L-arginine (300 mg/kg), L-NA (50 mg/kg), or L-NA plus L-arginine, twice (at 1000 and 1200 h). All animals were killed at approximately 1500 h.

In the third experiment, the OVX+E2 rats were divided into four groups, and each received icv injections of vehicle, antisense ODNs against nNOS, eNOS, or mNOS (1 µg/3 µl each) at 1300 h for 2 days before, and at 0800 h on the experimental day. All animals were decapitated at approximately 1500 h. The sequences of antisense ODNs for nNOS, eNOS, and mNOS are 5'-CGU UUC CAG UGU GCU CUU CA-3', 5'-CAG CCU UGG CAU CUU CUC CC-3', and 5'-GAG AAA CTT CCA AGG GCA-3', respectively. The sequences were adopted from a previous study (3) and were synthesized by a local company (DNAFax Inc., Taipei, Taiwan).

In the fourth experiment, both OVX+E2 and OVX+E2+P4 rats were used. The OVX+E2+P4 rats were further divided into three groups, and each received an injection of saline or L-NA (5 or 50 mg/kg) at 1000 and 1200 h. All the rats were decapitated at approximately 1300 h.

In the fifth experiment, both OVX+E2 and OVX+E2+P4 rats were used. The OVX+E2+P4 rats were further divided into four groups, and each received injections of the vehicle, L-arginine (300 mg/kg), L-NA (50 mg/kg), or L-NA plus L-arginine, twice (at 1000 and 1200 h). All animals were killed at approximately 1300 h.

In the sixth experiment, both OVX+E2 and OVX+E2+P4 rats were used. The OVX+E2+P4 rats were further divided into four groups, and each received icv injections of vehicle, antisense ODNs against nNOS, eNOS, or mNOS at 1300 h, for 2 days before, and at 0800 h on the experimental day. All animals were decapitated at approximately 1300 h.

Assay and statistical analysis
The level of DOPAC in the ME of each rat was determined by HPLC with electrochemical detection, which has been repeatedly reported (8, 9, 10, 13) and will not be repeated here. Serum PRL levels were determined by RIA using materials provided by the National Hormone and Pituitary Program of NIDDA, also as described (8, 9, 10, 13). The PRL for iodination was rat PRL-I-6, the standard was rat PRL-RP3, and the antibody was antirat PRL-S-7. The intra- and interassay coefficients of variance were 5% and 7%, respectively (n = 20).

Total nitrite/nitrate concentration in the serum was determined by using a nitrite/nitrate assay kit (Cayman Chemicals, Ann Arbor, MI), following the manufacturer’s instruction (18). Both nitrite and nitrate are stable metabolites of NO, and their level is commonly used as an estimate of NO synthesis. Briefly, plasma samples were ultrafiltered through a 10-kDa molecular mass cut-off filter (Schleicher & Schuell, Inc., Keene, NH) at 13,000 x g. Filtered plasma samples (in 40 µl) were loaded onto a 96-well plate and mixed with 10 µl of both enzyme cofactor and nitrate reductase. After incubation at room temperature for 3 h, 100 µl Griess reagent mix was added to each well. After 10 more min at room temperature, the absorbance of each well was determined on a microplate reader (BioTek Instruments, Inc., Winooski, VT; EL-311x) at 540 nm, and the nitrite/nitrate concentrations were deduced from a standard curve.

Either two-way (for Exp 1) or one-way ANOVA (for the rest) was used to test for significant differences among time points and/or treatments. One-way ANOVA, followed by the Student’s-Newman-Keuls’ multiple-range test, was performed for all groups. P < 0.05 was considered a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The role of NO in diurnal changes of TIDA neuronal activity and PRL surge in OVX+E2 rats
The OVX+E2 rats exhibited typical diurnal changes in their ME DOPAC and serum PRL levels: significant decreases of the former and increases of the latter at 1500 and 1700 h (P < 0.01; Fig. 1Go). Treatments of L-NA, twice (at 1000 and 1200 h) significantly prevented the decrease in ME DOPAC level and blunted the PRL surge (P < 0.01; Fig. 1Go).



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  		Figure 1. ME DOPAC and serum PRL levels, during the day, in OVX+E2 rats treated with or without L-NA (NA). Rats, OVX for 1 week and implanted with E2 (E)-containing capsules for 6 more days, were used. NA (50 mg/kg, ip) was injected twice (at 1000 and 1200 h on the experimental day), and the rats were decapitated at a specific time point. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. **, P < 0.01, compared with ME DOPAC or serum PRL levels at 1000 h; ##, P < 0.01, compared with the DOPAC or the PRL level of vehicle-treated OVX+E2 rats at the same time point.

 
The effects of L-NA on the 1500 h levels of ME DOPAC and serum PRL were confirmatory to the last experiment (P < 0.01; Fig. 2Go). Administration of L-arginine alone had no significant effect, whereas it significantly prevented the effects of L-NA when they were coadministered (P < 0.01; Fig. 2Go).



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  		Figure 2. ME DOPAC and serum PRL levels, at 1500 h, in OVX+E2 rats treated with NA and/or L-arginine (Arg). NA (50 mg/kg, ip) or Arg (300 mg/kg, ip) was injected twice (at 1000 and 1200 h) on the experimental day, and the rats were decapitated at approximately 1500 h. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. **, P < 0.01, compared with ME DOPAC or serum PRL levels of the vehicle-treated OVX+E2 rats; ##, P < 0.01, compared with the DOPAC or the PRL level of NA-treated OVX+E2 rats.

 
The effectiveness of L-NA on NO production was assessed indirectly by measuring the total nitrite/nitrate levels in the serum. Treatment of L-NA significantly decreased serum nitrite/nitrate levels [from 10.89 ± 1.02 µM in the control to 6.78 ± 0.67 µM (P < 0.01, n = 7)]. Treatment of L-arginine alone had a small (but nonsignificant) effect on the serum nitrite/nitrate level, i.e. to 12.55 ± 0.94 µM. However, L-arginine significantly prevented the L-NA-induced decrease of the serum nitrite/nitrate level, i.e. increased from 6.78 ± 0.67 to 9.25 ± 0.59 µM (P < 0.05, n = 7).

Pretreatment of antisense ODNs against messenger RNAs (mRNAs) of eNOS or nNOS effectively increased ME DOPAC levels and blunted the afternoon PRL surge at 1500 h (P < 0.01; Fig. 3Go). The same treatment of antisense ODN against mNOS, however, had no effect (Fig. 3Go).



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  		Figure 3. ME DOPAC and serum PRL levels, at 1500 h, in OVX+E2 rats pretreated with antisense ODNs against mRNAs for eNOS, nNOS, or mNOS. All rats received implants in their lateral cerebroventricle for icv injection 6 days before the experiment. The control group received vehicle injection as control, and the experimental groups received three injections (1 µg/3 µl each at 48, 24, and 7 h before decapitation) of antisense ODNs for eNOS, nNOS, or mNOS mRNAs; and all the rats were decapitated at approximately 1500 h. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. **, P < 0.01, compared with ME DOPAC or serum PRL level of the vehicle-treated OVX+E2 rats.

 
The role of NO in P4’s effect on TIDA neuronal activity and PRL secretion in OVX+E2 rats
A single injection of P4, at 0800 h, in OVX+E2 rats induced a significant decrease of ME DOPAC and increase of serum PRL levels at 1300 h, compared with those of vehicle-injected rats (P < 0.01; Fig. 4Go). Treatments of L-NA (twice, at 1000 and 1200 h) dose dependently (50, but not 5 mg/kg) prevented the effects of P4 (P < 0.01; Fig. 4Go).



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  		Figure 4. ME DOPAC and serum PRL levels, at 1300 h, in OVX+E2 and OVX+E2+P4 rats treated with two doses of NA. P4 (P, 2 mg/kg, sc) was given at 0800 h, NA (5 or 50 mg/kg, ip) was given twice (at 1000 and 1200 h) on the experimental day, and the rats were decapitated at approximately 1300 h. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. *, P < 0.05; **, P < 0.01, compared with ME DOPAC or serum PRL levels of OVX+E2 rat; ##, P < 0.01, compared with the DOPAC or the PRL level of vehicle-treated OVX+E2+P4 rats.

 
The effects of P4 and of L-NA on ME DOPAC and serum PRL levels at 1300 h were repeatedly observed (P < 0.01; Fig. 5Go). Administration of L-arginine had no effect by itself (Fig. 5Go); whereas it prevented the effect of L-NA when they were coadministered (P < 0.01; Fig. 5Go).



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  		Figure 5. ME DOPAC and serum PRL levels, at 1300 h, in OVX+E2 and OVX+E2+P4 rats treated with NA and/or Arg. P4 (P, 2 mg/kg, sc) was given at 0800 h, NA (50 mg/kg, ip) and Arg (300 mg/kg, ip) were given twice (at 1000 and 1200 h) on the experimental day, and the rats were decapitated at approximately 1300 h. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. **, P < 0.01, compared with ME DOPAC or serum PRL levels of vehicle-injected OVX+E2 rat; ##, P < 0.01, compared with the DOPAC or the PRL level of vehicle-treated OVX+E2+P4 rats; ++, P < 0.01, compared with the DOPAC and the PRL level of NA-treated OVX+E2+P4 rats.

 
The effects of P4 on ME DOPAC and serum PRL levels at 1300 h were repeatedly observed (P < 0.01; Fig. 6Go). Pretreatment of antisense ODNs for nNOS or eNOS mRNA was effective in preventing the effects of P4 (P < 0.01; Fig. 6Go); whereas that for mNOS was not (Fig. 6Go).



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  		Figure 6. ME DOPAC and serum PRL levels (at 1300 h) in OVX+E2 and OVX+E2+P4 rats treated with antisense ODNs against mRNAs for eNOS, nNOS or mNOS. All rats received implants in their lateral cerebroventricle for icv injection 6 days before the experiment. The control group received vehicle injection as control, and the experimental groups received three injections (1 µg/3 µl each at 48, 24, and 7 h before decapitation) of antisense ODNs for eNOS, nNOS, or mNOS mRNAs; and all the rats were decapitated at approximately 1300 h. Each bar is the mean of six to seven rats; the vertical line above each bar represents the SEM. **, P < 0.01, compared with ME DOPAC or serum PRL levels of vehicle-injected OVX+E2 rat; ##, P < 0.01, compared with the DOPAC or the PRL level of vehicle-treated OVX+E2+P4 rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The involvement of NO in the genesis of the E2-induced afternoon PRL surge is confirmatory to the previous finding (2). Nevertheless, our finding, that the effect of NO may act through the TIDA neurons, is novel. Inhibition of NO synthesis by L-NA or by antisense ODNs against eNOS or nNOS mRNA prevented the afternoon fall of TIDA neuronal activity, which is essential for the E2-induced afternoon PRL surge (8, 9). The significant decrease of the afternoon PRL surge in the same animals complemented well with the ME DOPAC data. Moreover, the finding that simultaneous injection of L-arginine negated the effects of L-NA further supports the role played by NO.

The other major finding of this study is that the advancing and potentiating effects of P4 on diurnal changes of TIDA neuronal activity and PRL secretion also involved NO. We have reported in a previous study (10) that P4 given at 0800 h on the experimental day can advance the rhythmic change of TIDA neuronal activity and PRL secretion by 1 h in OVX+E2 rats. The same findings were confirmed in this study. The effects of P4 were significantly prevented by using either a NOS inhibitor L-NA or one of the antisense ODNs against nNOS or eNOS mRNAs, indicating that NO is also involved in the action of P4. The finding that L-arginine could reverse the effect of L-NA further supports the notion.

The involvement of NO in the ovarian steroid-induced LH surge has previously been studied in more detail (1, 2, 3, 19). A removal of the inhibitory input of opioids (resulting in enhanced glutamatergic activity, and in turn, NO production and GnRH release) may underlie this event (19). On the other hand, the mechanism(s) for NO-stimulated PRL secretion is less understood. The present findings provide the first evidence that increased NO production may underlie the diurnal change of TIDA neuronal activity. Inhibition of NO production by a NOS inhibitor or antisense ODNs against mRNAs of eNOS or nNOS prevented the change. In most in vivo studies that used one or the other NOS inhibitor to block the NO synthesis (1, 2, 5, 6), however, the effectiveness of their action was justified by the end effects that were measured, e.g. hormone release and behavior, but not by direct determination of NO synthesis. Recent studies (20, 21, 22) have used extracellular level of cGMP in neuronal tissues sampled by microdialysis as an index for NOS activity. Serum or cerebroventricular fluid level of nitrite/nitrate has also been used (15, 16, 23). Although the latter index cannot provide direct evidence of the changes in NO in the hypothalamus, our data do indicate that treatment of L-NA significantly reduced NO synthesis in the rat and that coadministration with a large dose of L-arginine could nullify L-NA’s effect.

That NO can have an effect on central DA systems, mainly the nigrostriatal DA system, has been repeatedly reported (24, 25, 26, 27, 28, 29, 30), but with conflicting results. Studies using in vivo microdialysis or voltametry showed that NO may stimulate (24) or inhibit (25) DA release in the striatum, and NO may mediate the N-methyl-D-aspartate-induced DA release in the nucleus accumbens (26) or inhibit the N-methyl-D-aspartate-induced DA release in the striatum (27, 28). In vitro studies (29, 30) further showed that NO may inhibit the DA transporter function (hence, the DA uptake) in the striatal synaptosomes. A definitive role of NO in the control of striatal or accumbal DA is yet to be established.

A few studies (31, 32) have focused on the effect of NO on hypothalamic DA neurons. An in vitro study (31) showed that NO may inhibit the release, but not the synthesis, of DA in medial basal hypothalamic explants; and NO may inhibit the expression of tyrosine hydroxylase mRNA in the hypothalamic arcuate nucleus (32). The present findings also indicate that endogenous NO may have an inhibitory effect on the TIDA neuronal activity. Whether NO exerts its effect directly on TIDA neurons or indirectly through other neurotransmitters cannot be answered at present.

The diurnal change in TIDA neuronal activity has been shown to be a circadian rhythm that occurs on every day of the estrous cycle in female rats (8, 9). Though the presence of E2 is not essential for the occurrence of the rhythm, it increases the magnitude of the change. In fact, E2 has been shown to up-regulate NADPH-diaphorase staining and nNOS mRNA in the ventromedial nucleus and medial preoptic nucleus of the rat hypothalamus (33, 34, 35). The protein and mRNA levels of NOS in the hypothalamus may also increase during the proestrus afternoon (36). A recent study (37), using microdialysis, further showed that there is an ovarian steroid-independent diurnal rhythm of cGMP efflux in the medial preoptic area of female rats. The presence of E2 and P4 may shift the timing of the cGMP rhythm (37). This finding is compatible with ours (8, 9, 38), regarding the circadian nature of TIDA neuronal activity in female rats. We (39, 40) have also shown that several factors (e.g. acetylcholine, opioids, bombesin, etc.) are involved in the genesis of the TIDA rhythm. We (41) further reported that acetylcholine, opioids, and serotonin are also responsible for the action of P4 on advancing the rhythm. Thus, the production of NO may be one of the key links in the complex control of the rhythmic change in TIDA neuronal activity. The determination of their cause-effect relationships awaits future studies.

Our finding, that antisense ODNs against eNOS and nNOS mRNAs were effective in preventing the diurnal changes of ME DOPAC and serum PRL levels, is similar to an earlier study (3), in which treatments of the same sequences of antisense ODNs also prevent the LH surge. We already knew that both preovulatory LH and PRL surges in rats share some common neural control pathways (42, 43), as well as neurotransmitters (44, 45). We now add NO as a new common factor. Because mNOS is not produced in the hypothalamus under normal conditions, its antisense ODN was used as a control, as in the earlier study (3).

It should be noted that, although blocking the NO synthesis, using L-NA or antisense ODNs, was effective in preventing both changes in ME DOPAC and serum PRL, serum PRL levels were still higher than basal levels. This is consistent with our previous finding (39), that preventing the afternoon decrease of TIDA neuronal activity cannot completely block the PRL surge in OVX+E2 rats. Apparently, factors other than DA (e.g. the PRFs) also participate in the afternoon PRL surge. Without a prior decrease in TIDA neuronal activity to set the scene, however, only small (but not full) PRL surge can be induced by PRFs alone (46).

In summary, NO may play a significant role in the manifestation of endogenous TIDA rhythm in OVX+E2 rats and in the action of P4 on advancing the rhythm.


   Acknowledgments
 
We are grateful for the technical assistance of T. Y. Lee, S. L. Liang, and Z. F. Yuan. We also thank Dr. Y. J. Sung and J. C. Jea for helping with the nitrite/nitrate assay.


   Footnotes
 
1 This study was supported, in part, by Grants NSC86–2314-B010-M10 and NSC87–2314-B010–016 (to J.-T.P) from the National Science Council of the Republic of China. Back

Received July 21, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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