Nicotine Inhibits Pulsatile Luteinizing Hormone Secretion in Human

0021-972X/05/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 90(7):3908 –3913 Copyright © 2005 by The Endocrine Society doi: 10.1210/jc.2005-0041

Nicotine Inhibits Pulsatile Luteinizing Hormone Secretion in Human Males But Not in Human Females, and Tolerance to This Nicotine Effect Is Lost within One Week of Quitting Smoking Toshiya Funabashi, Akane Sano, Dai Mitsushima, and Fukuko Kimura Department of Neuroendocrinology (T.F., A.K., D.M., F.K.), Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan; and International University of Health and Welfare (F.K.), Tokyo 107-0062, Japan Context and Objective: Despite having increased knowledge of the adverse reproductive effects of smoking, it is unclear whether nicotine affects the pulsatile LH secretion in humans. We addressed this issue in male and female smokers and nonsmokers. Subjects and Methods: Twenty-nine male and 16 female nonsmokers and smokers were recruited as volunteers. In male smokers, nicotine effect was also studied before and after quitting smoking. In females, cyclic ovulatory function was assessed by measuring basal body temperature, and sampling studies were performed during the follicular phase. In the morning of the sampling day, an iv catheter was inserted into an anterobrachial vein, and blood samples (1.0 –1.5 ml each) were taken at 10-min intervals for 480 min, during which, at 240 min, nicotine was administered via a transdermal patch (Nicotinell transdermal therapeutic system) containing 17.5 mg nicotine.

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T HAS BEEN reported that sc injection of nicotine inhibits pulsatile LH secretion in ovariectomized rats (1). The site of this nicotine effect was assumed to be the medial basal hypothalamus because nicotine inhibited pulsatile LH secretion after partial or total deafferentation of the medial basal hypothalamus (1), but it did not inhibit GnRH-induced LH secretion in ovariectomized rats, ruling out its action on the anterior pituitary gland (1). More recently we have shown that nicotine inhibits the activity of the GnRH pulse generator in ovariectomized rats (2). Nicotine inhibited multiunit activity in the median eminence region of the hypothalamus that expresses characteristic increases intermittently in association with LH pulses in ovariectomized rats (3). We found a similar inhibitory effect of nicotine on the pulsatile GnRH secretion in the cultured rat embryonic olfactory placode (4). This latter in vitro study suggested that the inhibitory effect of nicotine on the GnRH pulse generator is mediated by the ␥-aminobutyric acid (GABA)A receptor system. Now we have focused our interest on the effect of nicotine on the pulsatile LH secretion in humans. The first aim of the present study (study 1) was to investigate whether nicotine First Published Online May 3, 2005 Abbreviations: CV, Coefficient of variation; GABA, ␥-aminobutyric acid; TTS, transdermal therapeutic system. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

Plasma LH was measured by immunoradiometric assay kits. Results: Nicotine significantly lengthened the interpulse interval of pulsatile LH secretion in male nonsmokers but not in female nonsmokers. In male smokers, nicotine did not lengthen the interpulse interval, and in female smokers it was also ineffective. After quitting cigarette smoking in male smokers, the refractory to nicotine effect disappeared within 1 wk. Conclusions: We conclude that nicotine inhibits pulsatile LH secretion only in males, and the tolerance developed to the nicotine effect disappears within 1 wk of quitting cigarette smoking. However, we cannot deny the possibility that nicotine effect would have been detected in females if more subjects had been studied. (J Clin Endocrinol Metab 90: 3908 –3913, 2005)

administered via a transdermal patch [Nicotinell transdermal therapeutic system (TTS); Novartis Pharma, K. K., Tokyo, Japan] affects the pulsatile LH secretion in male and female nonsmokers. To the best of our knowledge, there has been one study that examined the nicotine effect on the pulsatile LH secretion in female nonsmokers (5) but none in male nonsmokers. The second aim (study 2) was to determine how nicotine administered via transdermal patch affects the pulsatile LH secretion in male and female smokers. In this study we attempted to determine whether there was any change in the response to nicotine in comparison with that in nonsmokers. Although several reports have shown consistently, in male chronic smokers, an acute increasing influence of smoking on the secretion of GH (6 –9), prolactin (8, 10), and cortisol (ACTH) (6, 8, 9), either increasing influence (10) or no influence (6, 11) has been reported for LH. After reaching the conclusion in study 2 that the pulsatile LH secretion in male chronic smokers was refractory to nicotine, we further attempted (study 3) to determine whether quitting smoking changes the response to nicotine in male smokers. Subjects and Methods Subjects A total of 29 male and 16 female Japanese nonsmokers and chronic smokers were recruited as volunteers for these studies. We obtained written informed consent for the study from all volunteers and the

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Funabashi et al. • Nicotine Inhibits LH Secretion in Males

J Clin Endocrinol Metab, July 2005, 90(7):3908 –3913

approval of the study from the institutional ethics committee of the Yokohama City University School of Medicine. Subjects were questioned about general conditions of their health, and on the day of blood sampling, their blood pressure and electrocardiogram were examined to exclude cardiovascular diseases. All subjects were listed in Table 1 with indications to which study they were assigned. Subjects in study 1 were 11 male and eight female volunteers who had no history of smoking (nonsmokers). Subjects in study 2 were 18 male and eight female volunteers who had smoked daily for more than 6 yr (smokers). Brinkmann Index, i.e. the number of cigarettes per day ⫻ years (12), was shown in Table 1. Subjects in study 3 were first assigned to study 2 and then to study 3 if they agreed to quit smoking. Six of nine males succeeded in quitting smoking at least for 1 month TABLE 1. Summary of subjects

Subject no.

Age (yr)

Smoking duration (yr)

Study 1 Male nonsmokers (n ⫽ 11) 01 25 0 02 22 0 03 21 0 05 31 0 06 31 0 07 23 0 08 27 0 09 22 0 25 24 0 26 24 0 33 33 0 Female nonsmokers (n ⫽ 8) S9 24 0 S10 27 0 S11 21 0 S17 31 0 S21 26 0 S31 24 0 S33 25 0 S35 29 0 Study 2 Male smokers (n ⫽ 18) 13 24 6 14 26 7 15 22 7 16 21 6 17 24 6 18 24 8 19 29 10 20 22 7 21 21 7 22 24 10 23 24 6 24 29 11 30 24 7 34 24 7 35 22 7 38 28 8 42 23 7 49 28 13 Female smokers (n ⫽ 8) S6 24 7 S7 25 7 S8 28 9 S12 25 8 S13 25 9 S14 24 8 S41 27 7 S43 25 6

Brinkman Index

Pre

Time after quitting smoking 1W

0 0 0 0 0 0 0 0 0 0 0

* * * * * * * * * * *

0 0 0 0 0 0 0 0

* * * * * * * *

120 140 140 120 120 160 150 210 140 200 180 330 140 105 140 160 140 260

* * * * * * * * * * * * * * * * * *

140 105 90 160 180 120 105 60

* * * * * * * *

1M

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(Table 1). In female subjects, both nonsmokers and smokers, their cyclic ovulatory function was first assessed in a control cycle by measuring basal body temperature; they were all normally menstruating and have normal menstrual cycles (26 –35 d in length). There was no difference in the length of the menstrual cycle between nonsmokers (30.8 ⫾ 0.9 d) and smokers (29.6 ⫾ 0.5 d). The first day of the cycle was dated from the day when menstruation began, and sampling studies were performed on d 5–10, i.e. during the follicular phase.

Procedures All sampling studies were begun in the morning, between 0900 and 1000 h, in a quiet, temperature-controlled room. Subjects were permitted to take a breakfast 1 h before arriving but were instructed to consume, during the protocol, only blocks of a balanced food (Otsuka Pharmaceutical Co., Tokyo, Japan), each block containing 100 kcal for males and 50 kcal for females, at 60-min intervals and to drink green tea without sugar. This was to avoid a possible influence of changes in glucose level in blood caused by taking the meal (13). An iv catheter (LELCO Plus, Johnson-Johnson, Brussels, Belgium) was inserted into an anterobrachial superficial vein in the nondominant arm. Blood samples (1.0 –1.5 ml each) were taken through the catheter at 10-min intervals for a period of 480 min. After a 240-min control period, a Nicotinell TTS 10 (Novartis Pharma) containing 17.5 mg nicotine was attached to the abdominal skin and further blood samples were taken during another 240-min period (treatment period). Plasma samples were separated at room temperature and stored at ⫺20 C.

Analytical methods and statistical analysis

*

*

*

*

* *

*

* * * * *

*, Study to which subject was assigned. W, Week; M, month.

* * *

Plasma LH was measured with immunoradiometric assay kits (Daiichi Radioisotope Institute, Tokyo, Japan), and a total of 18 assays were done. The intra- and interassay coefficients of variation (CVs) estimated at the mean LH level of 8.6 mIU/ml were 4.0 and 10.0%, respectively. Plasma total testosterone levels in the first sample were measured by a commercial RIA at Otsuka Assay Laboratories (Tokushima, Japan). The LH pulse was identified using cluster analysis, a statistically based peak-detection algorithm (cluster 8) (14) with constant value of CV (8.5%). The parameters chosen were a nadir and peak size of 2 ⫻ 1 with a t statistic value of 2.6 for both increase and decrease. Because a last potential pulse during the 8-h blood sampling appeared to be not identified as a pulse by the cluster 8 program in many subjects, the last potential pulse was checked whether or not a LH pulse, using CV method (15, 16) in which a potential pulse was defined as an pulse when both the ascending and descending CVs were greater than 1.7 times the intraassay CV (15, 16). The CVs used for this pulse detection were 7.7–9.5%, depending on the assay CV. The analysis of LH pulsatility included the determination of interpulse interval (the time between the two neighboring pulses) and the pulse amplitude (difference between the peak and the prepeak nadir). The interpulse interval and the pulse amplitude were averaged for both the control period and the treatment period. If a first pulse appeared within 90 min in males and 60 min in females during the treatment period, this pulse was defined as a pulse during the control period because the average interpulse interval during the control period was 90 min in males and 60 min in females (see Fig. 2). One-way ANOVA followed by Fisher’s protected least significant difference post hoc comparison or Student’s t test was used to test the statistical significance in each group, and significance was accepted at P ⬍ 0.05.

Results Effects of nicotine on pulsatile LH secretion in male and female nonsmokers (study 1)

In male nonsmokers, secretion of LH was apparently pulsatile. Interpulse intervals varied among subjects, but the mean interpulse interval was approximately 90 min during the control period (Figs. 1 and 2). This was in good accord with previous reports (17, 18). As shown in Fig. 2, exposure to nicotine by attaching a Nicotinell TTS 10 significantly lengthened the interpulse interval (ANOVA, P ⬍ 0.05; post

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FIG. 1. Representative examples of effects of exposure to nicotine on the pulsatile LH secretion in male (left) nonsmokers and smokers, and female (right) nonsmokers and smokers. Nicotine was administered via a transdermal patch containing 17.5 mg nicotine (TTS 10). Shaded areas indicate the period of nicotine exposure. Numbers shown at the left upper position of each graph indicate the subject number. Triangles indicate statistically defined LH pulses.

hoc, P ⬍ 0.05), suggesting that nicotine inhibits the GnRH pulse generator activity in the hypothalamus, followed by inhibition of the pulsatile LH secretion from the pituitary. Exposure to nicotine did not affect the pulse amplitude in male nonsmokers (Table 2, ANOVA, P ⬎ 0.1). In female nonsmokers, pulsatile LH secretion with an interpulse interval of approximately 60 min was evident during the control period (Figs. 1 and 2). This interpulse interval in nonsmokers was in good accord with previous reports for the follicular phase (19 –21). In contrast to male nonsmokers, exposure to nicotine affected neither the interpulse interval (Fig. 2, ANOVA, P ⬎ 0.1) nor the amplitude (Table 2, ANOVA, P ⬎ 0.1). These results indicated that female nonsmokers were refractory to nicotine, in contrast to male nonsmokers. Effects of nicotine on pulsatile LH secretion in male and female smokers (study 2)

In male smokers, the LH secretory pattern was pulsatile and appeared to be similar to that in male nonsmokers dur-

ing the control period (Fig. 1). There was no significant difference in the interpulse interval during the control period between male nonsmokers and male smokers. The interpulse interval in male smokers during the treatment period was also not significantly different from that during the control period (Fig. 2, ANOVA, P ⬎ 0.5), indicating that exposure to nicotine did not affect the pulsatile LH secretion in male smokers. The same was true for the pulse amplitude (Table 2, ANOVA, P ⬎ 0.1). The results indicated that tolerance to the inhibitory nicotine effect on pulsatile LH secretion had been developed in male smokers. In female smokers, secretion of LH was pulsatile with an interpulse interval of approximately 60 min during the control period (Figs. 1 and 2). This interpulse interval was not significantly different from that during the control period in female nonsmokers (Fig. 2, ANOVA, P ⬎ 0.5). Furthermore, as in nonsmokers, exposure to nicotine did not affect the interpulse interval in smokers; there was no significant difference in the interpulse interval between the control period and the treatment period (Fig. 2, ANOVA, P ⬎ 0.5). The same was true for the pulse amplitude (Table 2, ANOVA, P ⬎ 0.1). TABLE 2. Amplitudes of LH pulses (mIU/ml)

Males Nonsmokers (n ⫽ 11) Smokers Before (n ⫽ 18) 1 wk (n ⫽ 9) 1 month (n ⫽ 6) Females Nonsmokers (n ⫽ 8) Smokers (n ⫽ 8) FIG. 2. Effects of exposure to nicotine on the interpulse intervals of the pulsatile LH secretion. Each bar and vertical line indicate the mean and SE, respectively. Numbers in parentheses indicate the number of subjects. *, P ⬍ 0.05.

Control period

Treatment period

2.57 ⫾ 0.32

3.00 ⫾ 0.74

NS

2.14 ⫾ 0.28 2.25 ⫾ 0.32 2.73 ⫾ 0.50

2.37 ⫾ 0.27 3.39 ⫾ 0.35 3.02 ⫾ 0.80

NS NS NS

1.70 ⫾ 0.28 1.82 ⫾ 0.19

2.14 ⫾ 0.55 2.26 ⫾ 0.32

NS NS

All the data are presented as the mean ⫾ SE. Before, 1 wk, and 1 month indicate before, 1 wk after, and 1 month after quitting cigarette smoking, respectively. NS, No significant differences were observed between control and treatment periods.



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Effects of nicotine on pulsatile LH secretion after quitting smoking in male smokers (study 3)

Representative profiles of the pulsatile LH secretion in smokers before and after quitting cigarette smoking are shown in Fig. 3. As already shown in study 2, exposure to nicotine did not have an inhibitory effect on the pulsatile LH secretion in male smokers, but this ineffectiveness of nicotine seemed to be lost after quitting smoking. Namely, at 1 wk and 1 month after quitting cigarette smoking, exposure to nicotine significantly lengthened the interpulse interval during the treatment period, compared with that during the control period (Fig. 4, ANOVA, P ⬍ 0.05; post hoc P ⬍ 0.05), without affecting the amplitude (Table 2, ANOVA, P ⬎ 0.1). Overall means of plasma concentrations of LH and testosterone in nonsmokers and smokers

To determine the influence of cigarette smoking on the LH secretion, overall means of serum LH concentrations were calculated for the control period in nonsmokers and smokers. As shown in Table 3, in males, mean plasma LH concentrations in smokers were significantly lower than in nonsmokers (Student’s t, P ⬍ 0.05), although there was no significant difference in plasma testosterone concentrations (Student’s t, P ⬎ 0.1) between nonsmokers (4.33 ⫾ 0.53 ng/ml, n ⫽ 11) and smokers (4.84 ⫾ 0.37 ng/ml, n ⫽ 18). In females, mean serum LH concentrations were not significantly different between nonsmokers and smokers (Student’s t, P ⬎ 0.1), in good accord with the ineffectiveness of nicotine on the parameter of pulsatile LH secretion. Discussion

Study 1 demonstrated for the first time that nicotine, which was administered via a transdermal patch, inhibits the pul-

FIG. 3. Representative examples of effects of exposure to nicotine on the pulsatile LH secretion in male smokers before and 1 wk (1 W) and 1 month (1 M) after quitting smoking. For more details, see Fig. 1.

FIG. 4. Effects of exposure to nicotine on the interpulse intervals of the pulsatile LH secretion before and after quitting cigarette smoking in male smokers. For more details, see Figs. 2 and 3.

satile LH secretion in male nonsmokers. The interval of pulsatile LH secretion was significantly increased after the administration of nicotine, with no significant changes in the amplitude. It was reported that the plasma concentration of nicotine obtained 4 h after the administration of a transdermal patch containing 19 mg nicotine, which was most similar to the dose contained in Nicotinell TTS 10, was at most 5 ng/ml (22). This dose was much lower than about 40 ng/ml seen about 5 min after the single cigarette smoking (23) or about 23 ng/ml seen 14 min during the cigarette-smoking period (10). Therefore, it is highly probable that cigarette smoking inhibits pulsatile LH secretion in male nonsmokers. Another important finding in the present study was that the inhibitory effect of a nicotine patch on the pulsatile LH secretion was not seen in female nonsmokers; Nicotinell TTS 10 (17.5 mg nicotine) did not inhibit the pulsatile LH secre-

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TABLE 3. Mean plasma LH concentrations during the control period Plasma LH (mIU/ml)

Males Nonsmokers Smokers Females Nonsmokers Smokers

4.23 ⫾ 0.11 (n ⫽ 11) 3.61 ⫾ 0.07 (n ⫽ 18)a 4.72 ⫾ 1.07 (n ⫽ 8) 4.68 ⫾ 0.55 (n ⫽ 8)

All the data are presented as the mean ⫾ a P ⬍ 0.01 vs. nonsmokers.

SE.

tion during the follicular phase of the menstrual cycle. Together with a previous report that cigarette smoking did not inhibit LH pulsatility in female nonsmokers at the late follicular phase (5), this result strongly suggests the presence of a sex difference in the response to nicotine, although we cannot deny the possibility that the nicotine effect would have been detected in females if more subjects had been studied. The mechanism for this sex difference was not ascertained in the present study, but it may be somehow related to differential roles of GABA in the control of pulsatile LH secretion between males and females. That is, for the control of pulsatile LH secretion, GABA neurons play a major role in male rats, mediating the negative feedback action of testosterone (24), whereas GABA neurons play only a minor role in female rats (25, 26) and opioid neurons mediate the negative feedback action of estrogen (27, 28). Taken together with our in vitro study suggesting that inhibitory nicotine effects on the GnRH pulse generator in the cultured rat embryonic olfactory placode appear via the stimulation of the release of inhibitory GABA (4) from GABA neurons existing in the same olfactory placode (26), the present result in male nonsmokers suggests that nicotine acts on GABA neurons to inhibit the activity of GnRH pulse generator present in the human hypothalamus (30). In study 2 done to observe nicotine effects on the pulsatile LH secretion in male smokers, neither the interval nor the amplitude of the pulsatile LH secretion responded to the nicotine patch, i.e. nicotine’s inhibition of the pulsatile LH secretion was no longer observed, suggesting that tolerance to hypothalamic actions of nicotine had developed in our chronic male smokers. In the previous studies done to see the effect of cigarette smoking on LH secretion in male smokers, nicotine was ineffective on LH secretion in two studies (6, 11) in accord with ours, whereas it increased the serum LH level within 14 min after starting cigarette-smoking period of 12 min (10). The reason for the difference between our result and the latter is obscure, but it probably relates to the fact that we used Nicotinell TTS 10 that elevates nicotine concentration in blood to only about 4 ng/ml, whereas the latter study used cigarette smoking that elevates the nicotine level rapidly to about 20 ng/ml. Although a considerable number of studies have dealt with the effect of smoking on male fertility, the available data do not conclusively demonstrate that smoking decreases male fertility (see review in Ref. 31). Sperm concentrations, motility, and/or morphology are often reduced, compared with results observed in nonsmokers, but they often remain within the normal range (see review in Ref. 32). Testosterone


levels in blood are either decreased (33, 34) or increased (35–37) in male smokers. Finally, LH levels in blood are not significantly different between male nonsmokers and smokers (34 –36). Thus, the present study is the first to demonstrate the decreased LH level in blood in male smokers, although testosterone levels were not changed. The reason previous studies did not find a decrease in male smokers may be related to the frequency of blood sampling from individual subjects: in two studies only one time and in another only three times. Because LH secretion is pulsatile, this sampling frequency may be too small to obtain the exact release level. Therefore, if deleterious effects of smoking on male fertility were discerned in male smokers, the decreased LH secretion may be a factor involved. However, the result should be interpreted with caution because the decrease in LH secretion was not associated with testosterone levels as stated above, even though a discrepancy between changes in LH levels and testosterone levels in smokers was evident in previous studies (35–37). Importantly, regulatory effects of LH on testosterone secretion are not simply related to the amount of LH or LH pulse frequency (38). A number of studies has demonstrated that nicotine induces tolerance to many of its acute effects in humans (39, 40). However, it has been controversial how quickly this chronic tolerance is lost after cessation of nicotine exposure, i.e. quitting smoking. There are reports describing a rapid (within several days) loss of tolerance to the cardiovascular effect of nicotine (41, 42). But 1 yr after quitting, tolerance to the dysphoric effect of nicotine was modestly maintained (43), whereas tolerance to the subjective and reinforcing effect of nicotine was completely lost (44), suggesting that tolerance loss may be response specific. However, more recently no clear loss of tolerance was observed in any cardiovascular, subjective, and reinforcing effects of nicotine 3 wk after quitting cigarette smoking (45). The present results showed that, in male smokers, tolerance to the inhibitory effect on LH secretion might disappear within 1 wk. We therefore suggest that tolerance to nicotine is relatively easy to disappear if pulsatile LH secretion is checked as a target of nicotine effects. Like female nonsmokers who showed no changes in pulsatile LH secretion in response to a transdermal nicotine patch, pulsatile LH secretion in female smokers also did not show responses to a transdermal nicotine patch. This seems somewhat surprising in light of reports that cigarette smoking has adverse effects on female fertility (31). However, as suggested previously (5), the antifertility effect of cigarette smoking in females may be mediated by mechanisms other than effects on the hypothalamus-pituitary system. For instance, cigarette smoking has been shown to reduce the ovarian reserve and lead to poor response to ovarian stimulation (29). However, because the latter authors documented that female smokers had a higher mean serum FSH concentration, the measurement of FSH in this series of studies may provide an answer. Acknowledgments Received January 7, 2005. Accepted April 25, 2005.


Address all correspondence and requests for reprints to: Fukuko Kimura, M.D., Ph.D., Department of Neuroendocrinology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawaku, Yokohama 236-0004, Japan. E-mail: [email protected]. ac.jp. This work was supported by a Smoking Research Foundation grant for biochemical research.

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