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Am J Physiol Heart Circ Physiol 276: H1281-H1288, 1999;
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Vol. 276, Issue 4, H1281-H1288, April 1999

Estradiol modulates vascular response to melatonin in rat caudal artery

Suzanne Doolen, Diana N. Krause, and Sue P. Duckles

Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697-4625


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to determine whether estrogen modulates the function of vascular melatonin receptors. We used the rat caudal artery and found that the contractile effects of melatonin were influenced by the estrous cycle, ovariectomy, and estrogen replacement. In arterial ring segments isolated from female rats, melatonin potentiated, in a concentration-dependent manner, contractions produced either by adrenergic nerve stimulation or by phenylephrine. Constrictor responses to melatonin were smaller in arteries from female rats in proestrus compared with other stages of the estrous cycle and after ovariectomy. Administration of 17beta -estradiol to ovariectomized female rats also resulted in decreased constriction of isolated arteries to melatonin; however, in vitro addition of 17beta -estradiol (10-7 M) had no effect. In the caudal artery, melatonin appears to act on two receptor subtypes that mediate contraction and relaxation, respectively. The selective melatonin MT2-receptor antagonist 4-phenyl-2-propionamidotetraline (4P-PDOT) enhanced constrictor responses to melatonin in arterial segments from intact female rats, consistent with the inhibition of MT2 receptor-mediated relaxation. In contrast, 4P-PDOT had no significant effect in arteries from ovariectomized female rats. However, when estradiol was replaced in vivo, the effect of 4P-PDOT on melatonin responses was restored. Thus circulating estradiol appears to enhance MT2 melatonin-receptor function in the thermoregulatory caudal artery of the female rat resulting in increased vasodilatation in response to melatonin.

melatonin receptor; estrous cycle; rat tail artery; estrogen replacement


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

COMPLEX INTERACTIONS APPEAR to exist between the pineal gland, its hormone melatonin, and the reproductive system and gonadal hormones. The relationship between the two endocrine systems has best been studied with regard to temporal regulation of reproduction, e.g., seasonal control of breeding and time of puberty onset (1, 2, 31, 33). Melatonin can inhibit gonadal function (33, 40), whereas estradiol inhibits melatonin production (18, 30-32, 39). The effects of melatonin are mediated in part by receptors in the brain and the pituitary (12, 13, 34, 35, 37, 42), and in part by peripheral actions (8, 21, 26, 36), although the latter are not well studied. Melatonin has been shown to suppress the expression of estrogen receptors in human breast cancer cells and in the hamster hypothalamus (17, 29). Little is known, however, about any possible reciprocal modulation of melatonin receptors by estrogen or about the effects of the two endocrine systems together on nonreproductive tissues. Given current interest in the human use of melatonin for treating jet lag, shift workers, and sleep disorders in the elderly (22), the potential interactions between melatonin and estrogen during puberty, childbearing years, and hormone replacement therapy after menopause (4) are relevant areas of study.

Melatonin receptors have been demonstrated in certain arteries by receptor autoradiography and functional responses (8, 14, 15, 21, 37, 38). In the rat caudal artery, a thermoregulatory blood vessel, melatonin potentiates constrictor responses to adrenergic nerve stimulation or alpha -adrenergic agonists in vitro (14, 15, 38). Our (8) recent study with this artery indicates that two opposing contractile responses to melatonin may coexist in vascular smooth muscle. Melatonin-induced vasoconstriction is enhanced in the presence of selective MT2 melatonin-receptor antagonists, suggesting that in addition to the constrictor effect, melatonin also produces relaxation that is mediated by MT2 melatonin receptors (8).1 The constrictor response exhibits a pharmacological profile that is most consistent with the melatonin mt1-receptor subtype; however, confirmation awaits the development of selective mt1 antagonists (8, 10, 21).

We (9) have noted differences in male and female vascular responses to melatonin. Recently, we observed that the effect of MT2 antagonists is more dramatic in female rat caudal arteries than in those arteries from male rats (unpublished observations). Consistent with these observations, levels of 2-[125I]iodomelatonin binding in rat caudal and cerebral arteries were found to differ not only between male and female rats but also to vary with the stage of the estrous cycle, after ovariectomy, and after estrogen replacement (37). The latter study suggests that estrogen modulates vascular melatonin receptors, although the ligand 2-[125I]iodomelatonin does not distinguish among the known melatonin-receptor subtypes (10, 11).

This study was undertaken to determine whether estrogen modulates the vascular effects of melatonin and if it does, which receptor subtype(s) are affected. The influence of female gonadal hormones on functional responses to melatonin was investigated in the rat caudal artery. Arteries were taken from intact female rats at different stages of the estrous cycle, as well as from ovariectomized rats with and without estrogen replacement. On the basis of our recent findings (8), we hypothesized that estrogen would modulate the MT2 receptor-mediated component of the contractile response to melatonin. Therefore, a selective inhibitor of the MT2-receptor subtype 4-phenyl-2-propionamidotetraline (4P-PDOT; 10-12) was also used to explore the influence of gonadal steroids on functional responses of the caudal artery to melatonin.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Three- to four-month old female Fisher 344 rats were housed under a 12:12-h light-dark cycle and fed ad libitum. The estrous cycle was determined in intact female rats by taking vaginal smears each morning by vaginal lavage. Smears were analyzed under a microscope to determine the type of cells present and the stage of the estrous cycle (23). Only female rats showing at least two consecutive 4- or 5-day estrous cycles were used. The established estrous cycle in each female was used to select the day of the experiment, at which time the estrous cycle stage was confirmed by vaginal smear.

Ovariectomy and estrogen replacement. Female rats 3-mo-old were anesthetized using ketamine (90 mg/kg body wt) and xylazine (10 mg/kg body wt) under aseptic conditions. Female rats were ovariectomized by making a small incision in the lower abdomen and removing both ovaries. In some female rats estrogen was replaced at the time of surgery by subcutaneously implanting 5 mm of Silastic tubing filled with granular 17beta -estradiol (6), which remained in place for 1 mo. We closed all wounds with surgical clips. Another group of ovariectomized female rats were given subcutaneous injections of 17beta -estradiol benzoate (50 µg/kg body wt) in safflower oil at 9 and 31 h before euthanasia. In all cases, ovariectomized rats were euthanized 4 wk after surgery. In some cases, trunk blood was collected after euthanasia and centrifuged at 3,000 rpm for 10 min. Approximately 0.5 ml of plasma was stored at -70°C to use later for determination of plasma levels of 17beta -estradiol by radioimmunoassay.

Tissue preparation. Animals were euthanized by decapitation on the morning of the experiment, 2-3 h after the lights were turned on. The caudal (tail) artery was quickly dissected out and placed in oxygenated Krebs buffer at 37°C consisting of (in mM) 5.15 KCl, 122 NaCl, 1.2 MgSO4, 11.5 glucose, 0.027 EDTA, 1.6 CaCl2, and 25.6 NaHCO3. Arterial segments (length 3 mm) were mounted on two platinum wires in a tissue bath filled with oxygenated Krebs solution (37°C). Isometric contractions were measured with Fort 10 transducers with MacLab analog-to-digital converter systems (World Precision Instruments, New Haven, CT). Tissues were equilibrated for 1 h and then stretched slowly to a resting tension of 0.8 g, previously determined to be the optimal resting tension for development of force. Tissues were rinsed once with Krebs solution before we started experimental protocols.

Experimental protocols. In studies of responses to adrenergic nerve stimulation, perivascular nerves were stimulated using two platinum electrodes placed 1 cm apart on either side of the arterial segment. Ten pulses of electrical stimulation were applied at low frequencies (1, 2, or 5 Hz). Tissues were allowed to rest for at least 2 min between stimulus trains. Once a consistent control response to transmural nerve stimulation (TNS) was achieved, one concentration of melatonin (10-10-10-7 M) was added to each arterial segment. After the addition of melatonin, a period of 5 min was allowed to pass before we resumed the nerve stimulation. The effect of melatonin was calculated as the percent increase in contractile response to TNS above the control contractile response to TNS determined in the absence of melatonin. In some experiments, this protocol was repeated in the presence of 17beta -estradiol. 17beta -Estradiol (10-7 M) was added 10 min before the determination of control responses to TNS and remained in the tissue bath throughout the subsequent experiment with melatonin.

In other experiments (14), the alpha 1-adrenergic agonist phenylephrine was used as a preconstrictor agent. Maximal contraction of each arterial segment was first determined by addition of 10-4 M phenylephrine. Tissues were then washed and precontracted to 10-20% of the maximum contraction by the addition of lower concentrations of phenylephrine (10-7-10-6 M). In the continued presence of phenylephrine, melatonin (10-10-10-5 M) was added cumulatively. In some experiments, the melatonin-receptor antagonist 4P-PDOT was added 5 min before the first addition of melatonin. The effect of melatonin was calculated as the change in tension (g) relative to the control contractile response to phenylephrine.

In some studies, the endothelium was removed by intimal rubbing. Endothelial integrity was assessed in each arterial segment by measuring relaxation to acetylcholine (10-6 M) after submaximal precontraction with norepinephrine (7 × 10-7 M).

Radioimmunoassay for plasma levels of 17beta -estradiol. Plasma samples were extracted using diethyl ether to isolate estrogens from potentially interfering substances, and then a double-antibody estradiol 125I radioimmunoassay kit (Diagnostic Products, Los Angeles, CA) was used according to manufacturer's instructions. Plasma 17beta -estradiol was measured by the ability of the sample to compete with labeled 17beta -estradiol for antibody sites. The detection limit of the assay was 1.4 pg/ml.

Materials. 4P-PDOT was purchased from Tocris Cookson (St. Louis, MO). Ketamine and xylazine were purchased from Western Medical Supply (Arcadia, CA). All other drugs were purchased from Sigma Chemical (St. Louis, MO). For the in vitro experiments melatonin (10 mM) was prepared in 20% ethanol; 4P-PDOT (0.1 mM) and 17beta -estradiol (0.1 mM) were prepared in 100% ethanol. All dilutions were made in Krebs buffer.

Statistical analysis. Data are means ± SE for each experimental group. Values for the magnitude of the response (Emax) and EC50 with 95% confidence intervals were calculated by nonlinear regression using the GraphPad Prism program. Unpaired Student's t-test or ANOVA with Newman-Keuls post hoc test was used to compare groups, including measurements for individual drug concentrations. P < 0.05 indicates statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of melatonin on responses to adrenergic nerve stimulation. Melatonin alone (10-10-10-5 M) had no effect on resting tone of caudal artery segments isolated from female rats. However, contractile responses to TNS were enhanced by melatonin (Fig. 1) as we (21) found previously using male rat caudal arteries. Melatonin had no effect on the duration of contractile peaks but significantly increased contractile force produced by adrenergic nerve stimulation at all frequencies tested.


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  		Fig. 1.   Effect of melatonin on contractile force in caudal artery segments from female rats in proestrus (A) or estrus (B). Representative tracings of contractile responses to transmural nerve stimulation (TNS) are shown. Each contraction was elicited by 10 pulses of TNS at 1, 2, or 5 Hz applied at times indicated (). Melatonin (3 × 10-9 M) was present in the tissue bath during the periods indicated.

We have shown that frequency-dependent responses of female rat caudal arteries to TNS are not significantly affected by the stage of the estrous cycle (23, 24). However, the ability of melatonin (3 nM) to potentiate TNS-induced contractions did vary during the female hormonal cycle; potentiation was significantly smaller during proestrus compared with the other days of the cycle (Figs. 1 and 2). No significant differences were observed among melatonin responses of arteries from animals in estrus, diestrus, and metestrus (Fig. 2); therefore, these arteries were evaluated together as one group, i.e., nonproestrus female rats. Circulating estrogen levels are relatively low in the nonproestrus stages of the estrous cycle but high in the proestrus stage (3).


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  		Fig. 2.   Influence of the estrous cycle on contractile effects of melatonin. Melatonin (3 nM) increased contractile responses to adrenergic nerve stimulation (TNS, 5 Hz) in caudal arteries obtained from female rats in estrus, diestrus, metestrus, and proestrus. Values are means ± SE. Percent increase above control contractions (TNS alone) is plotted for each group; n = 4-7 animals. * P < 0.05 compared with estrus, diestrus, and metestrus by ANOVA.

Concentration-response curves for melatonin also indicated significant differences between arteries from nonproestrus female rats and arteries from female rats euthanized on the morning of their proestrus day (Fig. 3). Arteries from ovariectomized female rats were also evaluated, and potentiation of contractile responses by melatonin was significantly greater than in arteries from proestrus female rats at all concentrations of melatonin tested (10-9-3 × 10-7 M). The effects of melatonin in arteries from nonproestrus or ovariectomized female rats were not significantly different from each other except at a melatonin concentration of 10-9 M. At this concentration, there was a striking enhancement of the constrictor effect of melatonin after ovariectomy.


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  		Fig. 3.   Concentration-response curves for melatonin in caudal artery segments from nonproestrus, proestrus, and ovariectomized (Ovx) female rats. Responses to TNS were elicited by 10-pulse stimulations at 5 Hz in absence (control) and presence of melatonin. Values are means ± SE for 7-14 rats. ** P < 0.05 compared with other two experimental groups.

Effect of acute in vitro treatment with 17beta -estradiol. The effect of melatonin on TNS-induced contraction in arterial segments from ovariectomized female rats was compared in the absence and presence of 17beta -estradiol (10-7 M) added in vitro. As shown in Table 1, potentiation by melatonin (10-10-10-7 M) was not significantly affected by the presence of 10-7 M 17beta -estradiol in the tissue bath. In vitro exposure to estradiol was varied from 10 min to 6 h; however, regardless of the exposure time period no effects were observed on vascular responses to melatonin.

                              
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  		Table 1.   Contractile effects of melatonin after exposure to 17beta -estradiol in vitro

Effect of in vivo treatment with 17beta -estradiol. To determine the effect of in vivo exposure to estradiol on vascular responses to melatonin, ovariectomized female rats were each given two subcutaneous injections of 17beta -estradiol benzoate (50 µg/kg body wt) at 31 and 9 h before euthanasia. As shown in Fig. 4, in vivo estrogen treatment decreased the ability of melatonin to potentiate TNS-induced contraction. The nature of the effect was frequency dependent. With 2 Hz of stimulation, estrogen treatment tended to shift the melatonin concentration-response curve to the right and significantly decreased the maximum effect (Fig. 4A). At the higher frequency (5 Hz), only responses to higher melatonin concentrations (>10-8 M) were significantly smaller in segments from estrogen-treated rats compared with those rats that were ovariectomized (Fig. 4B). In experiments where ovariectomized female rats were given only one subcutaneous injection of 17beta -estradiol benzoate 9 h before being euthanized, there was no effect on the functional response to melatonin (data not shown). In arteries from ovariectomized female rats given an injection of vehicle only, responses to melatonin were not different from those arteries of control ovariectomized animals (data not shown).


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  		Fig. 4.   Effect of estrogen treatment of Ovx female rats on vascular responses to melatonin in vitro. Treated animals were injected with 17beta -estradiol benzoate (50 µg/kg sc) at 31 and 9 h before euthanasia. Contractile responses of isolated caudal artery segments were elicited by a 10-pulse electrical stimulation at either 2 (A) or 5 Hz (B) in the presence and absence of melatonin. Values are means ± SE for 5-7 rats. * P < 0.05 compared with Ovx rats treated with 17beta -estradiol.

Effect of melatonin MT2-receptor antagonist. Our (8) recent data suggest that melatonin mediates two opposing responses in the caudal artery: contraction mediated by mt1-like receptors and relaxation induced by melatonin MT2 receptors. Thus the effects of gonadal hormones could be explained either by a decrease in constriction or by an increase in relaxation by melatonin. Currently, there are no selective mt1 antagonists available. However, 4P-PDOT is a selective antagonist of the MT2 melatonin-receptor subtype (10-12), and thus it was used to investigate these possibilities.

Because estrogen and/or melatonin may also affect the perivascular nerves, contractile responses to melatonin in this series of experiments were obtained after precontraction of the smooth muscle with the alpha 1-adrenergic agonist phenylephrine. As shown in Fig. 5, melatonin added cumulatively to tissues precontracted with phenylephrine (10-7-10-6 M) enhanced contractile force in a concentration-dependent manner, similar to our findings using TNS. The maximum response to melatonin was significantly smaller in arteries from intact female rats, which were taken at random throughout the estrous cycle, compared with ovariectomized female rats (Fig. 5, A and B; Table 2). Melatonin responses in arteries from ovariectomized female rats given estrogen replacement also tended to be smaller compared with arteries from ovariectomized female rats (Fig. 5, B and C). For example, the response of arteries from estrogen-replaced ovariectomized rats to 10-6 M melatonin was 0.15 ± 0.01 vs. 0.19 ± 0.02 g for ovariectomized rats, P < 0.05. The difference between the calculated Emax values for the two groups, however, did not reach significance (Table 2).


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  		Fig. 5.   Effect of 4-phenyl-2-propionamidotetraline (4P-PDOT; 10-7 M) on vascular responses to melatonin in caudal arteries precontracted with phenylephrine. Arteries were isolated from 3 groups of female rats: intact (A), Ovx (B), or Ovx receiving estrogen replacement (1 mo; C). Concentration-response curves for melatonin measured in presence and absence of 4P-PDOT (10-7 M) are shown. Values are means ± SE for 6-8 experiments. * P < 0.05 between control and 4P-PDOT-treated tissues.

                              
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  		Table 2.   Effect of 4P-PDOT on melatonin potency and maximal contractile response in caudal arteries from intact, ovariectomized, and estrogen-replaced ovariectomized rats

In arteries from intact female rats, 4P-PDOT significantly enhanced potentiation produced by higher melatonin concentrations (10-7-10-6 M; Fig. 5A; Table 2). In arteries from ovariectomized female rats, however, preincubation with 4P-PDOT (10-7 M) had no significant effect on potentiation by melatonin (Fig. 5B). However, after 17beta -estradiol was administered to ovariectomized female rats for 4 wk, 4P-PDOT significantly enhanced potentiation by melatonin at all concentrations tested (10-10-10-6 M; Fig. 5C; Table 2). In contrast to Emax, the EC50 of melatonin was unaffected by either 4P-PDOT or the estrogen status of the rat (Table 2).

Arterial segments used in the 4P-PDOT study showed little or no relaxation (12 ± 2%) in response to 10-6 M acetylcholine after precontraction with norepinephrine (7 × 10-7 M), which indicates a lack of functional endothelium in these segments. Plasma estradiol levels in ovariectomized rats used in the 4P-PDOT study were determined by radioimmunoassay. The plasma level of estradiol in ovariectomized female rats was 9 ± 3 pg/ml. In ovariectomized female rats treated with estrogen replacement, the plasma estradiol levels were 50 ± 11 pg/ml.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There is considerable evidence suggesting that pineal and reproductive hormones mutually interact, although in most cases the underlying mechanisms are not well understood (1, 2, 17, 29-33, 37, 39, 40, 42). The present study provides evidence that vascular reactivity to melatonin varies over the estrous cycle and is modulated by estrogen in vivo. The data support the hypothesis that circulating estradiol enhances melatonin MT2 receptor-mediated vasodilatation, suggesting one mechanism whereby female gonadal hormones influence the actions of melatonin.

In caudal arteries isolated from female rats, melatonin potentiated contraction produced either by electrical stimulation of perivascular adrenergic nerves or by activation of smooth muscle alpha 1-adrenoceptors. Melatonin has similar actions in male rat caudal arteries (14, 21, 38), and recent experimental evidence suggests more than one melatonin-receptor subtype (8, 27). The net vascular response to melatonin appears to be composed of two opposing responses: potentiation of contraction mediated by mt1-like melatonin receptors and relaxation mediated by MT2 receptors (8). This concept is further supported by recent demonstrations of mRNA for both mt1- and MT2-receptor subtypes in rat caudal artery smooth muscle cells (27). In the current study with female rats, the selective MT2-receptor antagonist 4P-PDOT enhanced constrictor responses to melatonin. These observations support the hypothesis that in both female and male caudal arteries activation of MT2 melatonin receptors produces relaxation in opposition to the predominant constrictor effects of melatonin.

The ability of melatonin to potentiate neurally evoked constriction was found to vary over the female rat estrous cycle. Contractile responses to melatonin were significantly smaller in caudal arteries obtained the morning of proestrus compared with those responses measured at other stages of the estrous cycle. Circulating levels of female gonadal hormones are elevated during proestrus but low during metestrus, diestrus, and estrus (3). When hormone levels were experimentally reduced by the removal of the ovaries, the magnitude of melatonin-induced constriction was similar to that of nonproestrus female rats. Together these data suggest that ovarian hormones influence the caudal artery response to melatonin. Estrogen is the most likely hormone candidate because its plasma concentration begins to rise dramatically on the evening before proestrus, whereas levels of progesterone and pituitary hormones such as the luteinizing hormone and follicle-stimulating hormone are elevated later in the proestrus day (3). The ability of melatonin to enhance vasoconstriction appears to be inversely correlated with estrogen levels, with the greatest potentiating effect of melatonin occurring when circulating estrogen is low, i.e., nonproestrus and ovariectomized states.

To further test this hypothesis, we investigated the effects of exogenous 17beta -estradiol using caudal arteries from ovariectomized rats. Although acute exposure to 17beta -estradiol in the tissue bath did not significantly affect contractile responses to melatonin, previous in vivo exposure of the rat to 17beta -estradiol did result in decreased constriction to melatonin in vitro. We observed this decrease with either nerve stimulation or phenylephrine precontraction and after estradiol administration either by subcutaneous injections or by Silastic tubing implants. With the latter method, plasma estradiol levels were found to approximate those measured in proestrus rats (3) and were significantly elevated compared with ovariectomized rats. These data further suggest that circulating estradiol modulates the vascular response to melatonin.

The most striking evidence that estradiol influences melatonin function is the effect of estrogen status on the effectiveness of the MT2-selective antagonist 4P-PDOT. In arteries from ovariectomized rats, i.e., with low levels of circulating estradiol, 4P-PDOT had no effect on smooth muscle responses to melatonin, which indicates that MT2 melatonin receptors produced no contractile response. However, in arteries from animals with higher plasma levels of estradiol, i.e., intact female rats and ovariectomized female rats treated with estrogen implants, 4P-PDOT significantly enhanced constrictor responses to melatonin. These findings suggest that after exposure to estradiol, the MT2 receptor elicits a functional response that can be blocked by selective antagonists. We hypothesize that estradiol alters contractile responses to melatonin by enhancing MT2 receptor-mediated vascular smooth muscle relaxation. This hypothesis is also consistent with our observations using another selective MT2-receptor antagonist 4-phenyl-2-acetamidotetraline, which enhanced melatonin-induced constriction to a greater extent in female rats than in male rats (unpublished observations).

Estradiol appears to enhance melatonin-mediated relaxation via a classic steroid action. According to this model, steroid hormones complex with specific cytosolic or nuclear receptors, which then bind chromatin resulting in regulation of gene transcription and ultimately of protein expression (5, 7). Estradiol also can have rapid actions on vascular tissue that are thought to result from nongenomic mechanisms (28). In our study, acute exposure of arterial segments to 17beta -estradiol for up to 6 h did not alter tissue responses to melatonin. In vivo administration of estradiol 9 h before euthanasia also had no effect. However, only longer in vivo exposures to estradiol (31 h to 1 mo) resulted in significant modulation of vascular responses to melatonin. Thus the actions of estradiol have a time course consistent with classic hormone regulation of protein expression. Our experiments cannot distinguish whether estradiol regulates melatonin-receptor expression directly or whether estradiol affects the transcription of another protein, which then indirectly alters melatonin-receptor function.

Estrogen alters the density and/or G protein coupling of a number of receptors (19, 20), and it may have a similar effect on melatonin receptors in vascular smooth muscle. The number of high-affinity 2-[125I]iodomelatonin binding sites in rat caudal and cerebral arteries was found to vary with gender and the female hormonal cycle and to be decreased by estrogen treatment compared with ovariectomized controls (37). The latter study suggests estrogen decreases melatonin-receptor expression by smooth muscle, which is opposite to what we would predict from the present results. Our functional data from endothelial-denuded arteries suggest that the number of smooth muscle MT2 receptors may instead be increased in the presence of circulating estradiol. 2-[125I]iodomelatonin autoradiography, however, does not appear to readily detect melatonin MT2 receptors (12, 25); furthermore, this agonist ligand does not distinguish between recombinant mt1 and mt2 receptors (10, 11, 34, 35). Therefore, the decrease observed in 2-[125I]iodomelatonin binding (37) could reflect inhibition of melatonin mt1-receptor expression by estrogen. If so, we would expect this effect to also decrease melatonin-induced vasoconstriction.

Thus more than one vascular mechanism may underlie the effects of estrogen observed in this study. Interestingly, we observed a unique effect of ovariectomy in the nerve stimulation experiments, i.e., responses to melatonin were increased at low concentrations and with low-stimulation frequencies. It is possible that melatonin has an action on the perivascular adrenergic nerves that is also influenced by estrogen. We have shown in rat caudal artery that ovariectomy decreases presynaptic inhibition by neuropeptide Y, which is most evident at low-stimulation frequencies (16). Melatonin has been shown to inhibit catecholamine release in the retina (11) and the hypothalamus (42), and we are currently investigating the possibility that a similar effect occurs in the caudal artery.

Estradiol is known to affect circulating melatonin levels via actions on the pineal gland (18, 30-32, 39). Therefore, it is possible that estradiol modulates melatonin responses in the caudal artery indirectly via regulation of pineal melatonin production. Circulating melatonin levels may regulate melatonin-receptor expression, although the evidence to date has come from studies measuring levels of 2-[125I]iodomelatonin binding (13). It is not known to what extent MT2 melatonin-receptor expression may be regulated by melatonin in vivo. Estrogen has many other actions, including direct modulation of vascular contractility by increasing nitric oxide-mediated relaxation (5-7). Although the latter action is not relevant for the endothelium-denuded artery segments used in the present study, we cannot rule out the possibility that other in vivo effects of estrogen contribute to the changes observed here in vascular responses to melatonin.

The caudal artery plays an important role in the thermoregulation of the rat (38, 41). Modulation of melatonin-receptor subtypes that mediate opposing contractile responses would be one way to regulate blood flow in the tail and optimize body temperature as melatonin levels rise and fall. This study provides evidence that estradiol enhances the MT2-mediated relaxation component of the net vascular response to melatonin. Such an action may serve to meet specialized thermoregulatory needs associated with reproductive processes, i.e., ovulation and changes in activity levels over the estrous cycle (37, 41). Estrogen-mediated alterations in melatonin-receptor subtypes may be an important mechanism underlying interactions between the pineal and reproductive systems. MT2 melatonin receptors have been localized in a variety of tissues including the brain and retina (11, 12, 34, 35). Thus it is possible that estradiol may modulate the function of MT2 melatonin receptors in other tissues as well.


    ACKNOWLEDGEMENTS

We thank Jonnie Sephus, Tom Nguyen, and Jeannie Truong for technical assistance; Dr. Greg Geary for assistance with the estrogen replacement protocol and animal surgeries; and Dr. Margarita L. Dubocovich for advice regarding MT2 antagonists.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-50775.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1  We use the official nomenclature and classification for melatonin receptors recently approved by the Nomenclature Committee for the International Union of Pharmacology (10). The denomination "mt1" corresponds to that of the recombinant melatonin-receptor subtype previously known as Mel1a (35). The "mt2" recombinant-receptor, previously known as Mel1b (34), is referred to here as "MT2" because it has been pharmacologically characterized in native tissue (11).

Address for reprint requests and other correspondence: S. P. Duckles, Dept. of Pharmacology, College of Medicine, Univ. of California, Irvine, Irvine, CA 92697-4625 (E-mail: spduckle{at}uci.edu).

Received 28 April 1998; accepted in final form 23 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alonso, R., P. Abreu, and N. Fajardo. Steroid influences on pineal melatonin production. In: Melatonin: Biosynthesis, Physiological Effects, and Clinical Applications, edited by H.-S. Yu, and R. J. Reiter. Ann Arbor, MI: CRC, 1993, p. 73-105.

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Am J Physiol Heart Circ Physiol 276(4):H1281-H1288
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