Sexually diergic hypothalamic-pituitary-adrenal axis responses to selective and non-selective muscarinic antagonists prior to cholinergic stimulation by physostigmine in rats.

Central cholinergic systems regulate the hypothalamic-pituitary-adrenal (HPA) axis differentially in males and females (sexual diergism). We previously investigated the role of muscarinic receptors in this regulation by administering physostigmine (PHYSO), an acetylcholinesterase inhibitor, to male and female rats pretreated with scopolamine (SCOP), a nonselective muscarinic antagonist. SCOP pretreatment enhanced adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses in both sexes, but males had greater ACTH responses while females had greater CORT responses. In the present study, we further explored the role of muscarinic receptor subtypes in HPA axis regulation by administering PHYSO to male and female rats following SCOP or various doses of either the M1 or the M2 selective muscarinic receptor antagonists, pirenzepine (PIREN) or methoctramine (METHO). Blood was sampled before and at multiple times after PHYSO. ACTH and CORT were determined by highly specific immunoassays. M1 antagonism by PIREN prior to PHYSO resulted in sustained, dose-dependent increases in ACTH and CORT: ACTH responses were similar in both sexes, and CORT responses were greater in females. M2 antagonism by METHO prior to PHYSO resulted in overall decreases in ACTH and CORT: ACTH and CORT responses were higher in females but lower in both sexes than the hormone responses following PIREN or SCOP pretreatment. Area under the curve analyses supported these findings. These results suggest that specific muscarinic receptor subtypes differentially influence the HPA axis in a sexually diergic manner.

Influence of environmental enrichment on hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine by osmotic mini-pumps, and nicotine withdrawal by mecamylamine in male and female rats.

In the present study, we determined the effects of environmental enrichment (EE; Kong Toys and Nestlets) on sexually diergic HPA axis responses to single-dose nicotine (NIC), single-dose NIC following continuous NIC administration for two weeks, and NIC withdrawal by single-dose mecamylamine (MEC) in male and female rats. Blood sampling occurred before and after MEC and NIC administrations for the determination of adrenocorticotropic hormone (ACTH) and corticosterone (CORT). Supporting and extending our previous findings, EE appeared to produce anxiolytic effects by reducing hormone responses: Male and female rats housed with EE had lower baseline ACTH and significantly lower HPA axis responses to the mild stress of saline (SAL) injection than did those housed without EE. The sexually diergic responses to single dose NIC, continuous NIC, and MEC-induced NIC withdrawal were reduced by EE in many male and female groups. ACTH responses to continuous NIC and MEC-induced NIC withdrawal were blunted to a greater extent in female EE groups than in male EE groups, suggesting that females are more sensitive to the anxiolytic effects of EE. Because EE lowered stress-responsive hormones of the HPA axis in most groups, EE may be a useful intervention for stress reduction in animal models of NIC addiction. As well, the effectiveness of EE in animal studies of NIC withdrawal may enlighten human studies addressing coping styles and tobacco cessation in men and women.

Sexually diergic hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine, continuous nicotine infusion, and nicotine withdrawal by mecamylamine in rats.

Hypothalamic-pituitary-adrenal (HPA) responses to single-dose nicotine (NIC) are sexually diergic: Female rats have higher adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses than do males. In the present study we determined HPA responses in male and female rats following single doses of NIC, a single-dose of NIC immediately following continuous NIC for two weeks, and NIC withdrawal by single-dose mecamylamine (MEC) following continuous NIC infusion for two weeks. Blood sampling occurred before and after MEC and NIC administrations for the determination of ACTH and CORT. In accordance with our previous findings, female ACTH and CORT responses to single-dose NIC were greater than male responses. This sex difference remained after single-dose NIC followed continuous NIC infusion, but HPA responses in both sexes were significantly lower in magnitude and duration than in the single-dose NIC alone groups. Sex differences also were observed following NIC withdrawal by MEC: the HPA responses to pretreatment with MEC were significantly higher in magnitude and duration in the continuous NIC groups than in the single-dose NIC groups. These results demonstrate that HPA responses to NIC are reduced and transient following continuous NIC infusion but are enhanced and sustained following NIC withdrawal by MEC after continuous NIC, suggesting that NIC habituation and withdrawal influence the stress responses in a diergic manner. These findings highlight the importance of sex differences in the effect of NIC on HPA axis activity and stress responsiveness, which may have implications for directing NIC-addiction treatment specifically towards men and women.

Sexually diergic, dose-dependent hypothalamic-pituitary-adrenal axis responses to nicotine in a dynamic in vitro perfusion system.

INTRODUCTION: The hypothalamic-pituitary-adrenal cortical (HPA) axis modulates physiological responses to stress. We previously reported sexually diergic, dose-dependent HPA responses in vivo following nicotine administration: Male rats had greater arginine vasopressin (AVP) responses than females, and female rats had greater adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses than males. The goal of the present study was to further investigate sexually diergic, dose-dependent HPA responses following nicotine addition to an in vitro model of the HPA axis, so that hormone output could be determined at each level of the axis. METHODS: Hypothalami, pituitaries, and adrenal glands were harvested from male and female rats. One-half hypothalamus, one-half pituitary, and one adrenal gland were placed individually into three jacketed tissue baths connected by tubing and perfused in series with physiological medium. Sampling ports between tissue baths were used to collect buffer before and after addition of various doses of nicotine, for measurement of AVP and corticotropin-releasing hormone (CRH) from the hypothalamus bath, ACTH from the pituitary bath, and CORT from the adrenal bath. Hormones were measured by highly specific immunoassays. RESULTS: Stable temperatures, flow rates, pH, and hormone baselines were achieved in the in vitro system. Consistent with our in vivo and earlier in vitro studies, nicotine added to the hypothalamus tissue bath significantly increased HPA responses in a sex- and dose-dependent manner: Males had greater AVP responses than did females, and females had greater CRH responses than did males. Sexually diergic ACTH and CORT responses were less apparent and were higher in females. DISCUSSION: Our in vitro system accurately models in vivo HPA responses to nicotine in both sexes and thus represents a reliable method for investigating the effects of nicotine on components of the HPA axis. These studies may be pertinent to understanding the biological differences to nicotine between men and women smokers.

Sequence of pituitary-adrenal cortical hormone responses to low-dose physostigmine administration in young adult women and men.

We previously demonstrated greater HPA axis activation in adult men compared to adult women following low-dose administration of the anticholinesterase inhibitor, physostigmine (PHYSO). Because blood sampling was done infrequently following PHYSO, the rise times of AVP, ACTH1-39, and cortisol could not be determined. In the present study, we determined the sequence of hormone increases by frequent blood sampling following PHYSO. Twelve adult women and 12 adult men underwent three test sessions 5-7 days apart: PHYSO, saline control, and repeat PHYSO. As in the earlier study, PHYSO produced no side effects in half the subjects and mild side effects in the other half, with no significant female-male differences. None of the hormone responses was significantly correlated with the presence or absence of side effects. In both women and men, the AVP increase preceded the ACTH1-39 increase, which in turn preceded the cortisol increase. The AVP and ACTH AUCs were significantly positively correlated in both women and men, supporting AVP as an acute stimulus to ACTH secretion. Also as in the earlier study, the AVP response to PHYSO was more than twice as great in men as in women, but the difference was not statistically significant. We therefore analyzed the results of both studies combined (N=26 women and 26 men). The men had a significantly greater AVP response and a trend toward a greater ACTH1-39 response compared to the women. These findings further support the concept of sexual diergism (functional sex difference) in the influence of CNS cholinergic systems on HPA hormone secretion.

Adrenal cortical responses to low- and high-dose ACTH(1-24) administration in major depressives vs. matched controls.

Increased hypothalamo-pituitary-adrenocortical (HPA) axis activity occurs in 30-50% of patients with major depression. This includes normal-to-increased adrenal cortical hormone (cortisol) secretion in spite of reduced corticotropin (ACTH) stimulation. A possible explanation is increased adrenal responsiveness to ACTH. Supporting this possibility is the finding of increased adrenal volume, which reverts to normal with successful treatment. Eight female and six male patients with major depression, and eight female and six male individually matched controls, underwent two test sessions 5-7 days apart. On the first day, a low ACTH(1-24) dose (0.014 microg/kg i.v.), equivalent to 1 microg in a 70-kg individual, was given. On the second day, a supramaximal stimulating dose (250 microg i.v.) was given. Serial blood samples were analyzed for immunoreactive (IR-)ACTH, ACTH(1-39), and cortisol. There were no significant sex or patient-control differences in IR-ACTH areas under the curve (AUCs) following low-dose ACTH(1-24), and the correlation between patient and matched control AUCs was +0.71, indicating good correspondence in the amount of circulating ACTH(1-24) available for adrenal stimulation. There were no significant sex or patient-control differences in cortisol response and no significant interaction between dose and subject group, indicating that patients did not differ from controls in their cortisol responses to either low- or high-dose ACTH(1-24). These findings do not indicate increased adrenal cortical responsiveness in patients with major depression. Neurochemical/neurohormonal and neural stimulatory factors other than ACTH might be responsible for the increased adrenal gland size and cortisol secretion, in spite of reduced pituitary ACTH secretion, that has been reported in this illness.

Growth hormone responses to low-dose physostigmine in elderly vs. young women and men.

BACKGROUND: Growth hormone (GH) secretion is a sensitive measure of CNS cholinergic neurotransmission, and GH decreases considerably with age. Cholinesterase inhibitors, which increase acetylcholine concentrations, have been used in elderly subjects to investigate the neuroendocrine effects of aging and Alzheimer's disease. However, there have been only a few studies of a potential sex difference in GH responses to cholinesterase inhibitors in elderly subjects, with mixed results. OBJECTIVE: We therefore administered low-dose physostigmine (PHYSO), a cholinesterase inhibitor, to normal, non-hormone-replaced, elderly women and men, to ascertain a potential sex difference in GH response. We hypothesized: (1) elderly women and men would have similar hormone responses, because of relatively low circulating estrogen in the women, and (2) the elderly women would have significantly lower baseline GH and GH responses to cholinergic challenge than the young women we studied previously. METHODS: Normal elderly women and men > or =65 years of age meeting stringent inclusion and exclusion criteria were studied on three test days, 4-7 days apart, by serial blood sampling for several hours for baseline GH, followed by administration of low-dose PHYSO (first and third days) or saline (second day) at 18:00 h. Frequent blood sampling was continued for several hours. Plasma GH and hypothalamo-pituitary-adrenal cortical hormones were measured in each sample. RESULTS: PHYSO administration produced no side effects in about half the elderly subjects and mild side effects in the other half, with no significant female-male differences and no significant relationship between the presence or absence of side effects and GH response. PHYSO significantly increased GH compared to saline, to a similar degree in the elderly women and men. The elderly women had a significantly greater GH response to PHYSO than did the young women, whereas GH responses were similar in the elderly and young men. CONCLUSIONS: These results indicate similar GH responses to low-dose PHYSO in elderly women compared to elderly men, and a significantly greater GH response in elderly women compared to young women. A likely mechanism is increased sensitivity of central cholinergic systems that inhibit somatostatin and/or enhance GHRH release from the hypothalamus.

Novel in vitro perfusion system for the determination of hypothalamic-pituitary-adrenal axis responses.

INTRODUCTION: The hypothalamic-pituitary-adrenal (HPA) axis is a three-gland component of the endocrine system and a key modulator of the stress response. We have developed a novel in vitro perfusion system to enable the study of pharmacological and hormonal challenges to tissue components of the HPA axis. In vivo studies have shown functional sex differences (sexual diergism) in HPA responses to cholinergic drugs, and in the present in vitro study, we examine these differences at several levels of the HPA axis. METHODS: Hypothalami, pituitaries, and adrenal glands were collected from male and female rats (n=3 per sex). One-half hypothalamus, one-half pituitary, and one adrenal gland were placed individually into three Erlenmeyer flasks connected by tubing. Flasks were perfused with medium (pH 7.4) at 37 degrees C. Sampling ports between the flasks were used to collect buffer for determination of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) release from the hypothalamus, pituitary, and adrenal flasks, respectively, over an extended baseline period, to determine stability of the system, and after nicotine administration. RESULTS: The perfusion system produced steady CRH, ACTH, and CORT baselines, the ACTH and CORT values being comparable to in vivo basal ACTH and CORT values in jugular-vein-cannulated rats. In vitro CRH, ACTH, and CORT responses to nicotine were significantly increased at 10 min and returned to baseline by 30 min, the CRH and ACTH responses from female tissues being greater than responses from male tissues. These sex differences were similar to those following nicotine administration in vivo. DISCUSSION: The ability of this novel, dynamic in vitro system to replicate in vivo HPA axis responses supports its potential as a new method for pharmacological and toxicological studies.

Rat estrous cycle influences the sexual diergism of HPA axis stimulation by nicotine.

We previously reported that female rats had significantly greater hypothalamic-pituitary-adrenal (HPA) axis responses to cholinergic stimulation by nicotine (NIC) than did male rats. Females in defined estrous cycle stages, however, were not studied because of sample size limitations. We further explored this finding by determining HPA axis responses to two doses of NIC in female rats (N = 101) during different estrous cycle stages, and in males (N = 69). NIC doses were: 0.3 mg/kg, which provided the greatest female-male difference in the earlier study, and 0.5 mg/kg, which stimulated the HPA axis similarly in the two sexes. Plasma AVP, ACTH, and corticosterone were measured. Proestrous and estrous females had higher ACTH responses to NIC (0.3 mg/kg) compared to metestrous and diestrous females, and compared to males. ACTH responses to NIC (0.5 mg/kg) were similar, regardless of estrous cycle stage or sex. Males had higher AVP responses to both NIC doses compared to females in all estrous cycle stages. Corticosterone responses followed the ACTH responses, except that females in all estrous stages started from a higher corticosterone baseline compared to males. These results are similar to our earlier findings across the estrous cycle with non-specific cholinergic stimulation by physostigmine and suggest that the nicotinic system contributes to the differential HPA axis responses to cholinergic challenge across the estrous cycle.

Environmental enrichment lowers stress-responsive hormones in singly housed male and female rats.

Structural and social aspects of an environmental system can influence the physiology and behavior of animals occupying that system. This study examined the physiological effects of environmental enrichment (EE) with Kong Toys and Nestlets on stress-responsive hormones of the hypothalamic-pituitary-adrenal (HPA) axis under basal and mild stress conditions in singly housed, jugular vein-cannulated, male and female rats. Animals of both sexes housed with EE had significantly lower baseline adrenocorticotropic hormone (ACTH) and corticosterone (CORT) concentrations compared to those housed without EE. ACTH responses to the mild stress of saline injection were significantly lower in female rats housed with EE. Interaction with the Kong Toys and Nestlets appears to have provided the rats with a diversion from monotonous cage life, resulting in lower HPA axis activity before and after mild stress. These results are important because low, stable baselines are essential for accurately discerning pharmacological and other influences on the HPA axis.

Plasma leptin suppression by arginine vasopressin in normal women and men.

Leptin inhibits appetite by activating several neuroendocrine systems, including the hypothalamo-pituitary-adrenal cortical (HPA) axis. In turn, elevated glucocorticoids can increase circulating leptin. We therefore measured plasma leptin in 12 normal women and eight normal men administered low-dose physostigmine (PHYSO) and arginine vasopressin (AVP) to stimulate the HPA axis. The subjects underwent four test sessions 5-7 days apart: PHYSO (8 microg/kg IV), AVP (0.08 U/kg IM), PHYSO + AVP, and saline control. Serial blood samples were taken before and after pharmacologic challenge and analyzed for leptin, adrenocorticotropin (ACTH)1-39, cortisol, and AVP. Estradiol and testosterone also were measured at each test session. PHYSO and AVP produced no side effects in about half the subjects and predominantly mild side effects in the other half, with no significant female-male differences. Correlations between side effects (absent or present) after PHYSO or AVP and the corresponding leptin responses were nonsignificant. Baseline plasma leptin concentrations were significantly higher in the women than in the men (p < 0.003). Leptin concentrations following PHYSO remained unchanged from baseline, indicating that the short-lived ACTH and cortisol increases produced by PHYSO did not affect leptin secretion. In contrast, AVP administration, while also increasing ACTH and cortisol, suppressed leptin, to a significantly greater degree in the women than in the men (p = 0.01). This significant suppression of leptin by AVP has not been previously described; physiologically, it may be part of a negative feedback regulatory system between central leptin and its activation of the HPA axis, by inhibition of leptin production or acceleration of its clearance.

Estrous cycle influences on sexual diergism of HPA axis responses to cholinergic stimulation in rats.

Central cholinergic systems differentially modulate hypothalamic-pituitary-adrenal (HPA) axis activity in female and male animals (sexual diergism). We previously reported that male rats had significantly greater HPA axis responses to stimulation by physostigmine (PHYSO), an acetylcholinesterase (AChE) inhibitor, compared to females. Females in defined estrous cycle stages, however, were not studied because of sample size limitations. We, therefore, determined HPA axis responses to stimulation by PHYSO in females during all estrous cycle stages (n = 78), and in male rats (n = 75). Plasma arginine vasopressin (AVP), adrenocorticotropic hormone (ACTH), and corticosterone (CORT) were measured. Estrous cycle stage was determined by light microscopy of vaginal smears. Proestrous and estrous females had higher ACTH and CORT responses compared to metestrous and diestrous females. Males had higher ACTH and AVP responses compared to females in all cycle stages. CORT responses followed the ACTH responses, except that females started from a higher baseline in all estrous stages, compared to males. These results suggest that cholinergic regulation of the HPA axis differs among females across stages of the estrous cycle, as well as between males and females. These effects are likely due to differences in circulating sex steroids and their actions within the brain.

Sexual diergism of hypothalamo-pituitary-adrenal cortical responses to low-dose physotigmine in elderly vs. young women and men.

We previously demonstrated that the reversible cholinesterase inhibitor, physostigmine (PHYSO), administered to normal young adult women and men (average age 35 years) at a dose that produced few or no side effects, resulted in a sex difference (sexual diergism) in hypothalamo-pituitary-adrenal cortical (HPA) axis responses: Plasma ACTH(1-39), cortisol, and arginine vasopressin (AVP) concentrations increased to a significantly greater extent in the men than in the women. To explore the effect of age on these sexually diergic hormone responses, in the present study we used the same dose of PHYSO (8 microg/kg IV) to stimulate ACTH(1-39), cortisol, and AVP secretion in normal elderly, non-estrogen-replaced women and elderly men (average ages 73 years and 70 years, respectively). The subjects underwent three test sessions 5-7 days apart: PHYSO, saline control, and a second session of PHYSO. Serial blood samples were taken for hormone analyses before and after pharmacologic challenge. As with the previously studied younger subjects, PHYSO administration produced no side effects in about half the elderly subjects and mild side effects in the other half, with no significant female-male differences. The hormone responses were 2-5 fold greater in the elderly subjects than in the younger subjects, but in contrast to the younger subjects, the elderly men did not have significantly greater hormone responses to PHYSO administration than did the elderly women. The ACTH(1-39) and AVP responses to PHYSO for the two sessions were significantly positively correlated in the men (+0.96, +0.91) but not in the women. None of the hormone responses was significantly correlated with the presence or absence of side effects in either group of subjects.These results indicate a greater sensitivity of the HPA axis to low-dose PHYSO, and a loss of overall sex differences in hormone responses, in elderly compared with younger subjects. The lack of a difference in side effects between the elderly women and men and the lack of significant correlations between presence or absence of side effects and hormone responses suggest that the increase in hormone responses with aging is due to correspondingly increased responsiveness of central cholinergic systems and/or the HPA axis, and not to a nonspecific stress response.

Sexual diergism of baseline plasma leptin and leptin suppression by arginine vasopressin in major depressives and matched controls.

Leptin inhibits appetite by activating several neuroendocrine systems, including the hypothalamo-pituitary-adrenal cortical (HPA) axis. In turn, chronically elevated glucocorticoids increase circulating leptin. HPA axis hyperactivity occurs in 30-50% of patients with major depression, but the few prior reports of leptin measurements in this illness have shown inconsistent results. We, therefore, measured plasma leptin in 12 female and 8 male unipolar major depressives and 12 female and 8 male individually matched normal controls administered low-dose physostigmine (PHYSO) and arginine vasopressin (AVP) to stimulate the HPA axis. The subjects underwent four test sessions 5-7 days apart: PHYSO (8 microg/kg IV); AVP (0-08 U/kg IM); PHYSO+AVP; and saline control. Serial blood samples were taken before and after pharmacologic challenge and analyzed for leptin, ACTH(1-39), cortisol and AVP. Estradiol and testosterone also were measured at each test session. PHYSO and AVP produced no side effects in approximately half the subjects and predominantly mild side effects in the other half, with no significant patient-control differences. Correlations between side effects (absent or present) after PHYSO or AVP and the corresponding leptin responses were non-significant in all groups. Baseline plasma leptin concentrations (mean+/-S.D.) were significantly higher in the female patients compared to the female controls (22.5+/-13.9 ng/ml vs. 12.3+/-9.7 ng/ml), whereas they were similar in the male patients and the male controls (3.9+/-1.4 ng/ml vs. 3.6+/-2.0 ng/ml). Leptin concentrations following PHYSO remained unchanged from baseline, indicating that the short-lived ACTH and cortisol increases produced by PHYSO did not affect leptin secretion. In contrast, AVP administration, while also increasing ACTH and cortisol, significantly suppressed leptin, more so in the women than in the men. Baseline leptin and the leptin decrease after AVP were moderately positively correlated with the Hamilton Depression Scale 'somatization' factor in the female patients (r=0.50) and more strongly correlated with the 'mood-depression' factor in the male patients (r=0.81). These findings indicate a sexual diergism (functional sex difference) in plasma leptin measures between major depressives and matched normal controls.