Endothelin-1 contributes to the sexual differences in renal damage in DOCA-salt rats

We investigated whether gender differences in renal damage in DOCA-salt hypertension are associated with effects of ovarian hormones and/or endothelin-1 (ET-1). Renal injuries and renal pre-pro-ET-1 mRNA expression were enhanced in male and female ovariectomized (OVX) DOCA rats versus female DOCA rats. Treatment with estrogen plus progesterone or progesterone, but not estrogen alone, attenuated renal damage and pre-pro-ET-1 mRNA expression in OVX DOCA rats. The ETA antagonist BMS182874 greatly ameliorated renal damage in male and OVX DOCA rats. In conclusion, the ovarian hormones have a protective role on the renal structural alterations in female DOCA rats by modulating effects of ET-1, via ETA receptors.     

Impairment of the L-arginine-nitric oxide pathway in mast cells from spontaneously hypertensive rats.

Serosal mast cells (MC) from 6 month old spontaneously hypertensive rats (SHR) were compared to MC from 6 month old Wistar Kyoto rats (WKYR) for their ability to release nitric oxide (NO). The relationship between histamine release and NO-like activity from these cells was also investigated. MC from SHR released less NO-like factor than MC from WKYR as assessed by the use of platelet aggregation and soluble guanylate cyclase activation as bioassays for NO. Sodium nitroprusside elevated the concentrations of cGMP to a similar extent in MC from SHR or WKYR. No changes in the levels of cAMP were observed. The release of histamine from MC induced by compound 48/80 or the calcium ionophore A23187 was greater in MC from SHR than in MC from WKYR. Thus, MC from SHR show a decreased production of NO-like activity which is reflected by a decreased ability to inhibit platelet aggregation. The decreased production of cGMP in the MC leads to an increased stimulated release of histamine.     

Ovariectomy Results in Variable Changes in Nociception,Mood and Depression in Adult Female Rats

Decline in the ovarian hormones with menopause may influence somatosensory, cognitive, and affective processing. The present study investigated whether hormonal depletion alters the nociceptive, depressive-like and learning behaviors in experimental rats after ovariectomy (OVX), a common method to deplete animals of their gonadal hormones. OVX rats developed thermal hyperalgesia in proximal and distal tail that was established 2 weeks after OVX and lasted the 7 weeks of the experiment. A robust mechanical allodynia was also occurred at 5 weeks after OVX. In the 5^th week after OVX, dilute formalin (5%)-induced nociceptive responses (such as elevating and licking or biting) during the second phase were significantly increased as compared to intact and sham-OVX females. However, chronic constriction injury (CCI) of the sciatic nerve-induced mechanical allodynia did not differ as hormonal status (e.g. OVX and ovarian intact). Using formalin-induced conditioned place avoidance (F-CPA), which is believed to reflect the pain-related negative emotion, we further found that OVX significantly attenuated F-CPA scores but did not alter electric foot-shock-induced CPA (S-CPA). In the open field and forced swimming test, there was an increase in depressive-like behaviors in OVX rats. There was no detectable impairment of spatial performance by Morris water maze task in OVX rats up to 5 weeks after surgery. Estrogen replacement retrieved OVX-induced nociceptive hypersensitivity and depressive-like behaviors. This is the first study to investigate the impacts of ovarian removal on nociceptive perception, negative emotion, depressive-like behaviors and spatial learning in adult female rats in a uniform and standard way.     

Effect of substance P on thyrotropin secretion from the pituitary gland in the rat

The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p less than 0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p less than 0.001), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10(-8) M and 10(-6) M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats.     

Prevention of adverse cardiac remodeling to volume overload in female rats is the result of an estrogen-altered mast cell phenotype

Previously, we have reported sex differences in the cardiac remodeling response to ventricular volume overload whereby male and ovariectomized (OVX) female rats develop eccentric hypertrophy, and intact (Int) female rats develop concentric hypertrophy. In males, this adverse remodeling has been attributed to an initial cascade of events involving myocardial mast cell and matrix metalloproteinase activation and extracellular collagen matrix degradation. The objective of this study was to determine the effect of female hormones on this initial cascade. Accordingly, an aortocaval fistula (Fist) was created in 7-wk-old Int and OVX rats, which, together with sham-operated (sham) controls, were studied at 1, 3, and 5 days postsurgery. In Int-Fist rats, myocardial mast cell density, collagen volume fraction, endothelin (ET)-1, stem cell factor (SCF), and TNF-α remained at control levels or were minimally elevated throughout the study period. This was not the case in the OVX-Fist group, where the initial response included significant increases in mast cell density, collagen degradation, ET-1, SCF, and TNF-α. These events in the OVX-Fist group were abolished by prefistula treatment with a mast cell stabilizer nedocromil. Of note was the observation that ET-1, TNF-α, SCF, and collagen volume fraction values for the OVX-sham group were greater than those of the Int-sham group, suggesting that the reduction of female hormones alone results in major myocardial changes. We concluded that female hormone-related cardioprotection to the volume stressed myocardium is the result of an altered mast cell phenotype and/or the prevention of mast cell activation.     

High rate of IgE-mediated histamine release from rat mesenteric mast cells.

IgE-dependent histamine release from rat mesenteric mast cells was investigated. Excised mesenterium was cut into pieces and incubated with IgE overnight at 4 degrees C for sensitization. Over 10 pieces of mesenterium specimen could be prepared from a rat. Antigen-induced histamine release from mesenterium specimen was initiated rapidly and reached a plateau in 5 min. In an optimal condition, over 50% of total histamine was released. In contrast, unpurified and purified peritoneal mast cells released only 22.5% and 5.3% of total histamine, respectively, upon IgE stimulation. Tranilast, a mast cell stabilizer, inhibited the histamine release from mesenteric mast cells significantly. The mesenterium might be useful material for studying tissue-associated mast cell activation.     

Differential regulation of the bumetanide-sensitive cotransporter (NKCC2) by ovarian hormones

The Na-K-2Cl cotransporter (NKCC2) regulates sodium transport along the thick ascending limb of Henle's loop and is important in control of sodium balance, renal concentrating ability and renin release. To determine if there are sex differences in NKCC2 abundance and/or distribution, and to evaluate the contribution of ovarian hormones to any such differences, we performed semiquantitative immunoblotting and immunoperoxidase immunohistochemistry for NKCC2 in the kidney of Sprague Dawley male, female and ovariectomized (OVX) rats with and without 17-β estradiol or progesterone supplementation. Intact females demonstrated greater NKCC2 protein in homogenates of whole kidney (334 ± 29%), cortex (219 ± 20%) and outer medulla (133 ± 9%) compared to males. Ovarian hormone supplementation to OVX rats regulated NKCC2 in the outer medulla only, with NKCC2 protein abundance decreasing slightly in response to progesterone but increasing in response to 17-β estradiol. Immunohistochemistry demonstrated prominent NKCC2 labeling in the apical membrane of thick ascending limb cells. Kidney section NKCC2 labeling confirmed regionalized regulation of NKCC2 by ovarian hormones. Localized regulation of NKCC2 by ovarian hormones may have importance in controlling sodium and water balance over the lifetime of women as the milieu of sex hormones varies.     

The recovery of the neurally evoked secretory response of rat colonic mucosa after irradiation is independent of mast cells

The ability of the enteric submucosal plexus to influence the transport of water and electrolytes in the colon was investigated in rats for 1 week after acute whole-body gamma irradiation. The involvement of neuroimmune links in the epithelial responses to nerve stimulation was confirmed by the sensitivity of the tissue to tetrodotoxin, mepyramine and doxantrazole. At 1 and 3 days after irradiation, colon tissues were hyporesponsive to nerve stimulation. This was associated with a drastic diminution of mucosal mast cell numbers, tissue histamine levels, and rat mast cell protease II (RMCP II) levels, and by a decreased maximal epithelial response to exogenously added histamine. The responses to electric-field stimulation were insensitive to both mepyramine and doxantrazole. At 7 days, neurally evoked responses recovered, despite the virtual absence of mast cells, tissue histamine and RMCP II, and the continuing decreased response to histamine. The responses were insensitive to doxantrazole but were decreased by mepyramine. This study showed that the establishment of a normal epithelial response to neural stimulation can occur despite the radiation-induced depletion of mucosal mast cells. The recovery of the epithelial response, which was sensitive to mepyramine, may be ascribed to the reappearance of an unknown histaminergic pathway, which probably has indirect effects on epithelial transport but is independent of nerve-mast cell connections.     

A potential role for catecholamines in the development and progression of carcinogen-induced mammary tumors: hormonal control of beta-adrenergic receptors and correlation with tumor growth.

In order to gain further knowledge on the beta-adrenergic receptor system in DMBA-induced rat mammary tumors, we have studied the correlation between changes in tumoral beta-adrenergic receptor concentration and distribution, progesterone receptor status and tumor growth after ovariectomy and treatment with various ovarian and adrenal steroids, or induction of hyperprolactinemia. Autoradiographic localization of beta-adrenergic receptors in ovariectomized (OVX) animals shows very weak labeling with [125I]cyanopindolol. In these tumors, the connective tissue is predominant, while the epithelial cell content is very low. Similarly, when direct measurements of [125I]cyanopindolol are performed with membrane preparations, beta-adrenergic receptor concentration is sharply reduced 2-3 weeks following ovariectomy or treatment with LHRH against [D-Trp6, des-Gly-NH2(10)]LHRH ethylamide. This effect on the beta-adrenergic receptor population in the tumor is accompanied by the well known effect of castration on tumor growth and progesterone receptor levels, namely a marked regression of tumor growth and a significant decrease in progesterone receptor concentration. Treatment of OVX rats with 17 beta-estradiol (E2) alone or in combination with progesterone (P) caused a highly significant increase in beta-adrenergic and progesterone receptor levels, as well as tumor growth. A similar sharp increase in the value of the three parameters studied was observed following daily treatment of OVX rats with dehydroepiandrosterone (DHEA) or androst-5-ene-3 beta,17 beta-diol (5-ene-diol). The autoradiographic localization of beta-adrenergic receptors in OVX rats treated with 5-ene-diol showed that the epithelial cells were numerous with a high degree of labeling. On the other hand, treatment of OVX animals with the androgen dihydrotestosterone (DHT) did not produce significant changes in beta-adrenergic receptor levels or tumor growth. Finally, endogenously-induced hyperprolactinemia by implanting three anterior pituitary glands under the kidney capsule of OVX animals resulted in a significant increase in beta-adrenergic and progesterone receptor levels as well as tumor growth. The positive correlation observed between changes in beta-adrenergic receptor concentration, progesterone receptor levels and tumor growth indicates a high sensitivity of the beta-adrenergic receptor population of DMBA-induced rat mammary tumors to the hormonal milieu, and suggests that the beta-adrenergic receptor system may represent a valuable parameter of hormone responsiveness.     

Effects of estradiol and progesterone on body composition, protein synthesis, and lipoprotein lipase in rats

Prior studies suggest that estradiol and progesterone regulate body composition in growing female rats. Because these studies did not consider the confounding effect of changes in food intake, it remains unclear whether ovarian hormones regulate body composition independently of their effects on food intake. We utilized a pair-feeding paradigm to examine the effects of these hormones on body composition. In addition, skeletal muscle protein fractional synthesis rate and adipose tissue lipoprotein lipase activity were measured to examine pathways of substrate deposition into fat and fat-free tissue. Female Sprague-Dawley rats [pubertal: 7-8 wk old; 190 +/- 0.5 (SE) g] were separated into four groups: 1) sham-operated (S; n = 8), 2) ovariectomized plus placebo (OVX; n = 8), 3) ovariectomized plus estradiol (OVX+E; n = 8), and 4) ovariectomized plus progesterone (OVX+P; n = 8). All ovariectomized groups were pair-fed to the S group. Body composition was measured using total body electrical conductivity. The relative increase in fat-free mass was greater (P < 0.01) in the OVX group (31 +/- 2%) than in the S (17 +/- 2%), OVX+E (18 +/- 2%), and OVX+P (22 +/- 2%) groups. The fractional synthetic rates of gastrocnemius muscle protein paralleled changes in fat-free mass: OVX had a higher (P < 0.05) synthesis rate (21 +/- 3%/day) than S (12 +/- 2%/day), OVX+E (11 +/- 2%/day), and OVX+P (8 +/- 1%/day) groups. Body fat increased in the S group (31 +/- 7%; P < 0.01), whereas the OVX groups lost fat (OVX: -10 +/- 7%; OVX+E: -15 +/- 7%; OVX+P: -13 +/- 7%). No differences in lipoprotein lipase were found. Our results suggest that estradiol and progesterone may regulate the growth of fat and fat-free tissues in female rats. Moreover, ovarian hormones may influence skeletal muscle growth through their effects on skeletal muscle protein synthesis.     

Long-term caffeine-induced inhibition of EAC cell progression in relation to gonadal hormonal status

Inhibitory action of caffeine (a tri-methylxanthine alkaloid) on progression or pathogenesis of lung, breast and ovarian cancer including Ehrlich ascites carcinoma (EAC) cell development has been reported. This information led the authors to study the effect of long-term administration of caffeine (20 mg/kg/day; po for 22-27 consecutive days) on the development of EAC cells in relation to serum gonadal hormones (LH, FSH, 17-OH-beta-estradiol (E2) and progesterone) in adult Swiss albino female mice. Measurement of gonadal hormones in serum using RIA showed that (a) long-term caffeine treatment significantly increased LH (except for 27 consecutive days) and decreased FSH (except for 24 and 27 consecutive days) and both E2 and progesterone (except for 22 and 24 consecutive days) levels, (b) development of EAC cell for 10-15 days, significantly increased LH but decreased FSH, E2 and progesterone levels and (c) long-term caffeine consumption during the development of EAC cell (i) restored the EAC cell- or caffeine-induced induction of LH and reduction of FSH level to their normal levels and (ii) withdrew/reduced the EAC cell-induced reduction in only E2 but not progesterone level. These results therefore, suggest that prolonged caffeine exposures may inhibit the development of EAC cell through the reduction or restoration of EAC cell-induced disruption of ovarian hormonal status to their normal status via the modulation of Hypothalamic-Pituitary-Gonadal (HPG) axis.     

Antigenic stimulated release of arachidonic acid, lipoxygenase activity and histamine release in a cloned murine mast cell MC9

A cloned murine mast cell MC9 expresses phospholipase and lipoxygenase activity when stimulated with IgE and hapten. Addition of DNP-BSA to sensitized MC9 cells causes release of 58% of the cell histamine and 127 pmoles LTC4/10(6) cells. Prelabelling studies with [1-14C]-arachidonic acid showed that LTC4 production was proceeded by the release of arachidonic acid from membrane phospholipids. Approximately 8.7% of the cell arachidonic acid was released and half of this was converted to LTC4. The remaining radioactivity was converted to diHETES including LTB4 (15%), 5-HETE (10%), free arachidonic acid (10%), reesterified 5-HETE and arachidonic acid (8%) and prostaglandins (7%). This stimulation was dependent on hapten (DNP-BSA) and extracellular Ca++. Under identical conditions the calcium ionophore A23187 stimulated the release of 10.3% of the total cell arachidonic acid, and 51% of this was metabolized to LTC4. In addition the ionophore stimulated the release of 61% of the total cellular histamine.     

Prolactin receptor expression by splenocytes from rats in various hormonal states

Prolactin (PRL) is mitogenic for lymphocytes in vitro , but the responsiveness of lymphocytes depends on the in vivo hormonal status of the rats from which the cells were obtained. Lymphocytes from ovariectomized (OVX) rats, but not from rats in oestrus or from male rats, respond to prolactin; administration of oestradiol to OVX rats diminishes the response. In order to determine if a correlation exists between lymphocyte responsiveness to prolactin and levels of cell surface prolactin receptors (PRL-R) expression, the percentage of splenocytes and each splenocyte subpopulation expressing surface PRL-R from rats of various hormonal states (OVX, oestradiol-injected OVX, oestrus and male) was analysed by single-colour and dual-colour flow cytometric analysis. We found that approximately 20% of splenocytes expressed surface PRL-R regardless of hormonal states ( n =16). The majority (85%) of PRL-R positive splenocytes were B lymphocytes whereas 11.1% and 4.8% of splenocytes expressing the PRL-R were CD4 positive T-helper (T_H) and CD8 positive T-cytotoxic (T_C) lymphocytes, respectively. B lymphocytes also stained more brightly than T lymphocytes. This distribution of PRL-R expression did not show significant alterations on total splenocytes or T_H and T_C lymphocytes during various hormonal stages. However, the percentage of PRL-R-positive B lymphocytes increased markedly in OVX rats (twofold), compared to rats at oestrus. In summary, no correlation was found between the responsiveness to prolactin as a mitogen and levels of PRL-R expression by lymphocytes from rats at different hormonal states. This result suggests that sex steroid hormones may control prolactin responsiveness of lymphocytes by affecting the signal transduction pathway through PRL-R rather than by altering the level of the cell surface receptor expression.     

17Beta-estradiol inhibits ovariectomy-induced expression of inducible nitric oxide synthase in rat aorta in vivo

The objective of this study was to elucidate a role of ovarian steroid hormones in the production of immunologic nitric oxide (NO) synthases in the female rat aorta in vivo. Aortic homogenates were analyzed by using western blot with isoform-specific antibodies against endothelial NOS (eNOS) and inducible NOS (iNOS). Two weeks after ovariectomy (OVX), rats (10-week-old) were treated with 17beta-estradiol (E2) and/or progesterone (P4) for 5 days, and aortae were obtained from these rats on the following day. OVX markedly increased the levels of iNOS protein in abdominal aorta, whereas treatment with E2 or a combination of E2 and P4 inhibited the induction of iNOS in the aorta. The present findings indicate that endogeneous estrogen negatively regulates the expression of iNOS in abdominal aorta, and suggest that changes in the levels of circulating estrogen may affect vascular function.     

Involvement of the corticoadrenal hormones in the pheromonal restoration of reproductive activity in aging rats

The involvement of the adrenal progesterone and corticosterone in the early gonadotropin secretion associated with the pheromonal restoration of ovarian cyclic activity (PRCA) in aging female rats is studied. PRCA is induced by male urinary pheromones and is preceded by an alpha-adrenergic-mediated release of the hypothalamic decapeptide luteinizing hormone-releasing hormone and plasma increases of estradiol, progesterone and the gonadotropins luteinizing hormone and follicle stimulating hormone. Aging reproductive Wistar female rats were used to study the effects of bilateral adrenalectomy and of a subcutaneous injection of the antisteroid RU486 on plasma levels of corticosterone, progesterone and gonadotropins in rats stimulated with nasal spraying of male urine (MU) or saline. The results demonstrate that progesterone and corticosterone released by MU are from adrenal origin, and that these adrenal secretory products are critical for MU-induced increase of gonadotropins. This suggests that olfactory stimulation of ACTH release stimulates adrenal release of progesterone and corticosterone, and both trigger the events that initiate the activation of the hypothalamus-pituitary-ovarian axis that leads to PRCA.     

Age-related alterations in pulsatile luteinizing hormone release: effects of long-term ovariectomy, repeated pregnancies and naloxone

Aging of the female reproductive system may be regulated by changes at the hypothalamic, pituitary, and ovarian levels. Long-term ovariectomy (LT-OVX) and/or multiple pregnancies delay age-related deterioration of several parameters of reproductive potential in rodents. We tested whether long-term suppression of cyclic ovarian hormone release that is normally associated with the 4- to 5-day estrous cycle decelerates age-related decreases in the frequency of luteinizing hormone (LH) pulses to assess whether hormonal milieu influences the rate of aging of the pulse generator. We determined the percentage of rats exhibiting pulsatile LH secretion, mean LH levels, and amplitude and frequency of LH pulses in seven groups of ovariectomized (OVX) rats. Young (3-4 mo), middle-aged (8-10 mo), and old (18-22 mo) virgin rats, ovariectomized 4 wk (4WK-OVX) prior to experimentation, were used to determine the effect of age. The effect of long-term ovarian hormone deprivation was tested by ovariectomizing rats at 2-3 mo of age and using them when they were middle-aged (8-10 months) or old (18-22 mo). The effect of deprivation of cyclic increases in ovarian hormones associated with repeated estrous cycles was tested by using retired breeder (RB) rats that had been ovariectomized 4 wk prior to experimentation. Each rat was implanted with a right atrial cannula and bled the next day at 10-min intervals for 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroids and hypothalamic norepinephrine release: studies using in vivo brain microdialysis

This study employed microdialysis in urethane-anesthetized female rats to monitor ovarian steroid-dependent changes in KCl-evoked levels of extracellular norepinephrine (NE) in the ventromedial hypothalamus. An initial KCl stimulus (Sl) increased NE from low or undetectable levels in all animals. A second KCl stimulus (S2) given several hr later evoked 40% less NE release than did Sl in ovariectomized (OVX) females or OVX females given only estrogen or progestin. In contrast, the two KCl-evoked NE releases were equivalent in OVX females administered both estrogen and progestin. These results suggest that ovarian steroids may act as presynaptic modulators of NE release in the ventromedial hypothalamus.     

Estrous Cycle Dependent Fluctuations of Regulatory Neuropeptides in the Lower Urinary Tract of Female Rats upon Colon-Bladder Cross-Sensitization

Co-morbidity of bladder, bowel, and non-specific pelvic pain symptoms is highly prevalent in women. Little evidence is present on modulation of pelvic pain syndromes by sex hormones, therefore, the objective of this study was to clarify the effects of hormonal fluctuations within the estrous cycle on regulatory neuropeptides in female rats using a model of neurogenic bladder dysfunction. The estrous cycle in female rats (Sprague-Dawley, 230–250 g) was assessed by vaginal smears and weight of uterine horns. Neurogenic bladder dysfunction was induced by a single inflammatory insult to the distal colon. Protein expression of calcitonin gene related peptide (CGRP), substance P (SP), nerve growth factor (NGF), and brain derived neurotrophic factor (BDNF) in the pelvic organs, sensory ganglia and lumbosacral spinal cord was compared in rats in proestrus (high estrogen) vs diestrus (low estrogen). Under normal physiological conditions, concentration of SP and CGRP was similar in the distal colon and urinary bladder during all phases of the estrous cycle, however, acute colitis induced a significant up-regulation of CGRP content in the colon (by 63%) and urinary bladder (by 54%, p≤0.05 to control) of rats in proestrus. These changes were accompanied by a significant diminution of CGRP content in L6-S2 DRG after colonic treatment, likely associated with its release in the periphery. In rats with high estrogen at the time of testing (proestrus), experimental colitis caused a significant up-regulation of BDNF colonic content from 26.1±8.5 pg/ml to 83.4±32.5 pg/ml (N = 7, p≤0.05 to control) and also induced similar effects on BDNF in the urinary bladder which was also up-regulated by 5-fold in rats in proestrus (p≤0.05 to respective control). Our results demonstrate estrous cycle dependent fluctuations of regulatory neuropeptides in the lower urinary tract upon colon-bladder cross-sensitization, which may contribute to pain fluctuations in female patients with neurogenic bladder pain.     

Ovarian steroidogenic responsiveness to exogenous gonadotropin stimulation in young and middle-aged female rats

Reproductive aging in the female rat is associated with gradual declines in LH secretion and ovarian progesterone (P) production. This study examined whether the influences of aging on P levels reflect decreased ovarian responsiveness to gonadotropin stimulation, as opposed to changes in gonadotropin release. Young and middle-aged regularly cyclic female rats received sodium pentobarbital to block endogenous proestrous luteinizing hormone (LH) surges, followed by administration of various doses of human chorionic gonadotropin (hCG). Similar treatments were performed in middle-aged acyclic persistent-estrous (PE) females. Injection of hCG resulted in equivalent plasma hCG levels in each treatment group. At the lowest hCG dose tested, a significant rise in plasma P levels was observed in middle-aged cyclic rats, but not in young cyclic or middle-aged PE females. This unexpected finding may reflect accelerated follicular development in middle-aged cyclic females, as suggested by a previous study. At the intermediate dose, young and middle-aged cyclic but not PE rats displayed significantly increased P in response to hCG. At the highest dose tested, all three groups of rats displayed increased P levels after hCG stimulation. However, P concentrations were significantly lower in middle-aged PE than regularly cyclic females. Northern and slot blot hybridization analyses revealed that ovarian mRNA levels for cytochrome P450 side-chain cleavage, the rate-limiting enzyme in P synthesis, were markedly reduced in PE rats following hCG stimulation. These findings indicate that ovarian responsiveness to gonadotropin stimulation is impaired in middle-aged PE, but not regularly cyclic rats, and suggest influences of cycle status on the biochemical and molecular mechanisms regulating ovarian steroid production. Furthermore, these findings reveal that attenuated P production in middle-aged proestrous rats is due to attenuated preovulatory LH surges, rather than decreased ovarian sensitivity to LH.     

Subcellular localization of brain mast cell histamine in developing rat

The intracerebroventricular administration of compound 48/80 or polymixin B to rats 0 to 60 days old, produced a decrease both in the histamine which sediments in the crude nuclear fraction, as well as in the number of mast cells in the brain. In contrast, the histamine-releasers did not affect histamine levels in subcellular fractions where neuronal histamine is found. Once released, histamine disappeared rapidly (t 1/2 = 3.8 min). In untreated animals and in those treated with histamine releasers, the number of mast cells/g in the whole brains of developing rats and in the cerebral regions of adult rats showed a close correlation with the histamine levels in the crude nuclear fraction. The content of histamine per mast cell in adult rat brain was estimated to be about 13 pg/cell. Histologic examination of the subcellular fractions revealed the presence of intact mast cells in the crude nuclear fraction obtained from untreated animals, and of degranulated mast cells in the same fraction obtained from animals treated with histamine releasers. The mast cell contribution to adult rat brain histamine levels was about 22%. Our results strongly support that most of the histamine which sediments in the crude nuclear fraction of the rat brain is located in mast cells. Determination of histamine in the crude nuclear fraction and in the supernatant of this fraction is proposed as an easy way for identifying the cellular pool altered by any treatment affecting brain histamine levels.