In general, when an organism is placed under sudden stress, the level of activity of the cells exciting complement of enzyme increases, leading to a spurt in production. Epinephrine synthesis in the adrenal medulla is also subject to the biochemical influence of .the adrenal cortex. Because blood from the cortex passes through the medulla, most of the chromaffin cells are exposed to high levels of cortical steroids, When the steroids are absent, it has been found that the enzyme that catalyzes the conversion of norepinephrine into epinphrine is degraded at an unusually high rate, leading to the decline of its cellular level. Thus, the intimate anatomical relation of the cortex and the medulla extends to the interaction at molecular level as well.

Stress is known to induce the secretion of hormones from the adrenal cortex as well as from the meulla. The cortical hormones apparently ensure that the synthesis of epinephrine is maintained¹⁾. Kohler and his collaborators²⁾ found that estradiol and progesterone administration to intact female rats increased adrenal tyrosine hydrosylase activity. Moreover, tyrosine hydroxylase activity is known to be increased significantly in diethylstilbesterol-induced pituitay tumors of the rats than in control pituitaries. These data support the possible existence of local catecholaminergic mechanism(s) that could be modulating prolactin secretion from pituitary tumors and from ectopic pituitaries³⁾.

In hypothalamic and vocal control nuclei of male zebra finches, all hormone-induced changes in noradrenergic neurotrasmission are estrogen-dependent, while modulation of dopaminergic function is much more variable and frequently androgen-dependent⁴⁾. Steroids are also known to affect catecholamine (CA) synthesis⁵⁾, degradation⁶⁾, release⁷⁾ and re-uptake⁹⁾.

However, the steroid-induced hyperpolarization in guinea-pig ganglion neurons in vitro was accompanied by a change in the input resistance of the cell, indicating an involvement of some kind(s) of ion channel(s) in the action of glucocorticoid¹⁰⁾. It has been also shown that brief hyper-polarization and increased potassium conductance are produced by 17-β-estradiol in rat medial amygdala brain slices¹¹⁾. Moreover, according to the results of Miguel and his co-workers¹²⁾. estradiol seems to decrease the ability of the adrenal medulla to release CA to the peripheral blood, whereas progesterone most affects tyrosine hydrosylase activity. Being that its effects temporally and partially depend on estrogens.

It has been also found that 17-α-estradiol inhibits DMPP- and high K⁺-induced CA release from the cat adrenal glands and 45Ca⁺⁺ uptake into bovine chromaffin cells in culture stiulated by DMPP or high K⁺¹³⁾. More recently. Laret and his collaborators¹⁴⁾ have suggested that the amygdaloid catecholaminergic systems exert an inhibitory effect on CA content of the adrenals and the ovary, and influence the ovarian estradiol secretion mechanism. This influnce of the amygdala in the regulation of the ovary could be mediated by an efferent pathway from the central nervous system to the ovary.

Thus, there are many controversial reports on CA release from the adrenal medulla. The present study was designed to examine the effect of 17-α-estradiol on CA secretion from the isolated perfused rat adrenal glands and to clarify the mechanism of its action.

In the present study, the experimental results suggest strongly that 17-α-estradiol causes the inhibitory effect of CA secretion evoked by cholinergic (nicotinic or muscarinic) receptor stimulation and Bay-K-8644, but not that by membrane-depolarization, indicating that this effect may be mediated by inhibiting the influx of extracellular calcium into the rat adrenomedullary chromaffin cells through the activation of inhibitory estrogen receptors, and it also play a modulatory role in regulating CA secretion.

In support of this idea, Lopez and his collabolators¹³⁾ have found that the steroid hormone 17-α-estradiol blocks CA secretion from adrenal chromaffin cells in response to some secretagogues. Although this effect is especially clear upon nicotinic stimulation and, to a lesser extent with high K⁺ stimulation, it is dubious in the case of muscarinic-mediated secretion. However, it seems that there is some difference between results of previous reports and the present investigation. In the present experimental results, 17-α-estradiol markedly inhibited CA secretory responses of DMPP and McN-A-343, but did not affect that of excess K⁺. The resson for this discrepancy is not explained, but the discrepancy may be due to the species differences in the adrenal medulla.

Moreover, Leret and his co-workers¹⁴⁾ have also shown that, in the adrenal medulla, the CA levels measured in animals lesioned with 6-hydroxydopamine were significantly higher than those of the controls. Thus, it could be suggested that the CA of the amygdala plays an inhibitory role in animals with 6-hydroxydopamine lesions in the CNS region. The activity of the adrenal medulla can also be influenced by steroid hormones produced in the ovary²⁹⁾. These hormones not only modify the production of corticosteroids but also the activity of some medullary enzymes. Namely, it has been demonstrated that low levels of ovarian steroids reduce MAO and phenylethanolamine-N-methyltransferase (COMT) activities, with no changes of COMT activity¹⁴⁾. This mechanism could be involved in the increase of CA in the adrenal medulla obtained after lesions in the amygdala. A similar effect has also been described in several brain areas^30–32). Thus, the adrenal medulla activity of COMT, the enzyme involved in the inactivation of CA by o-methylation, was significantly increased after the treatment with either estradiol or progesterone²⁹⁾.

Miguel and his co-workers¹²⁾ have found that the CA release, measured after in vitro incubations of adrenomedullary tissue, is significantly reduced during the estrosus phase. Most likely, pre-ovulatory increase of estradiol, occurring during the early progestrous³³⁾, would be responsivle for this decreased CA release, since the pharmacological administration of estradiol to overiectomized rats produced a similar decrease in the CA release 24 hr after the steroid injection. They have also suggested that the effects of sex steroid on adrenal medulla would be produced by a direct action on chromaffin cells through a classic genomic mechanism³⁴⁾. In this respect, the existence of cytoplasmic steroid receptors has been observed in the whole adrenal³⁵⁾.

However, these results are not concordant with the present experimental data. Because, first, the inhibitory effect by 17-α-estradiol of CA release is established within 20 min and, second, its effect is readily reversible upon washing out the hormone. Rather, its effect might be ascribed to the direct action to the membrane rapid actions observed in different neuronal systems. The rapid effect that, in the present investigation, 17-α-estradiol inhibited CA secretion evoked by DMPP and McN-A-343 is not explained in the frame of the classical genomic mechanism of steroid hormone actions through activation of high-affinity cytosolic receptors and their further translocation to the cell nucleus to induce new protein synthesis. These effects are generally known to take hours or even days to appear³⁶⁾.

Rather, the catecholamine blocking effects are better explained in the newly emerging concept of cell membrane effects of steroids in neurons. In support of these ideas, there are several recent example reports of these nongenomic, rapid effects of steroids. By applying steroids directly to specific neurons, immediate changes in their firing frequency are observed^10,11,37,38). The activity of hormone-sensitive striated muscles can be enhanced within minutes of steroid treatment³⁹⁾. As these rapid steroid effects are not prevented by protein synthesis inhibitors^11,39), it seems that they result from a direct action of steroids on the plasma membrane. In the light of these facts, it is suggested that the target for 17-α-estradiol is located on the plasma membrane of the rat adrenomedullary chromaffin cells. If this hormone were acting at some intracellular site on the secretory machinery, the responses to all secretagogues would be blocked the same extent.

Furthermore, in the present study, 17-α-estradiol also inhibited the secretory effect of CA evoked by Bay-K-8644, which is known to be a Ca⁺⁺uptake²¹⁾ and CA release²²⁾. In terms of this view, the present finding that 17-α-estradiol depressed CA secretory effects by Bay-K-8644, as well as cholinergic receptor-stimulation without effect on membrane-depolarization, strongly suggests that the inhibitory effect of 17-α-estradiol is mediated through the blockade of Ca⁺⁺ entry into the chromaffin cells.

In support of these results, there is now sizeable literature demonstrating a key role of Ca⁺⁺ influx through voltage-sensitive Ca⁺⁺ channels as a physiological pathway for activation of adrenal CA^40–45). Moreover, it is found that the activation of nicotinic receptors stimulates CA secretion by increasing Ca⁺⁺ entry through receptor-linked and/or voltage-dependent Ca⁺⁺ channels in both perfused rat adrenal glands⁴⁶⁾ and isolated bovine adrenal chromaffin cells^47–49), and that the muscarinic receptor activation causes an increase in adrenal CA secretion independent of extracellular Ca⁺⁺ in various species^50–52) and in cytosolic free Ca⁺⁺ in bovine isolated adrenal chromaffin cells without associated CA secretion^45,53–55).

However, recently, Lim and Hwang⁵⁶⁾ have found that both DMPP and McN-A-343 greatly cause CA secretion from the isolated perfused rat adrenal medulla by a calcium-dependent exocytotic mechanism. Considering in connection with these findings, the present results that 17-α-estradiol inhibited CA releasing responses induced by DMPP and McN-A-343 as well as that by Bay-K-8644, but did not that by excess K⁺, indicate strongly that this inhibitory activity may be mediated by inhibiting Ca⁺⁺ influx into chromaffin cells through receptor-linked and/or voltage-sensitive Ca⁺⁺ channels. However, the fact that 17-α-estradiol did not affect CA secretion evoked by high K⁺ could not be clearly explained, because DMPP and high K⁺ are known to increase Ca⁺⁺ uptake into chromaffin cell through the recruitment of voltage-sensitive Ca⁺⁺ channels^28,57). In addition, Lopez and his co-workers¹³⁾ have also observed that 17-α-estradiol depresses the secretory responses of CA evoked by both DMPP and high K⁺ from the perfused bovine and cat adrenal medulla.

In terms of these reports, the present result that 17-α-estradiol fails to inhibit CA release induced by excess K⁺ from the rat adrenal glands might explain its high potency in blocking CA secretion evoked by DMPP or McN-A-343 than by excess K⁺, indicating that there is species difference.

On the other hand, in this study, the fact that the inhibitory effect of 17-α-estradiol to the secretory responses evoked by DMPP and McN-A-343 as well as by ACh was recovered to the considerable extent of the control in the presence of tamoxifen indicates strongly that this hormone causes depression in CA release evoked by cholinergic (nicotinic and/or muscarinic) receptor stimulation through the activation of inhibitory estrogen receptors on chromaffin cell membrane. Tamoxifen is a competitive inhibitor of estrogen binding at the estrogen receptor^24–25) and blocks estrogen action in breast cancer cells which contain receptors. Recently, Wiseman⁵⁸⁾ has described that tamoxifen protects membranes and lipoprotein particles against oxidative damage, and that this antioxidant action is likely to contribute to the observed cardio-protective action of tamoxifen and supports the use of this compound in treating and even preventing breast cancer. Membrane-mediated mechanisms of tamoxifen action, through a putative modulation of membrane fluidity are likely to play an important role in it anti-cancer action and its ability to reverse multi-drug resistance and could also lead to clinical uses as anti-candida and anti-viral agents.

Also, in the present investigation, 17-β-estradiol, an isomer of estradiol, inhibited CA secretion evoked by cholinergic stimulation. In support of this fact, after 3 weeks of ovariectomy in female Clarias batrachus, CA levels were elevated significantly in the preparatory, pre-spawinig and spawning phases and were ulaltered in the post-spawning season²³⁾.

Administration of 17-β-estradiol to this fish resulted in both seasonal and differential effects on the CA levels. Moreover, it has been reported that 17-β-estradiol also produces brief hyperpolarization and increased potassium conductance in rat medial amygdala brain slices¹¹⁾.

Besides, the finding that 17-α-estradiol-induced inhibitory effects were unchanged by co-administration with metoclopramide, which is known to block dopaminergic receptors, leading to hypertensive crisis⁵⁹⁾ and CA release by calcium-dependent exocytotic mechanism⁶⁰⁾, suggests that the inhibitory effects are not associated with dopaminergic receptors in the rat adrenal glands.

In conclusion, 17-α-estradiol causes the inhibition of CA release evoked by cholinergic (nicotinic and/or muscarinic) stimulation as well as that by Bay-K-8644, but not that by membrane-depolarization from the isolated perfused rat adrenl glands, indicating strongly that this inhibitory effect may be mediated by inhibiting calcium influx into the chromaffin cells through the activation to the inhibitory estrogen receptors and it also plays a modulatory role in regulating CA secretion.