Immunohistochemical localization of oxytocin receptors in human brain.

The neuropeptide oxytocin (OT) regulates rodent, primate and human social behaviors and stress responses. OT binding studies employing (125)I-d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)] ornithine vasotocin ((125)I-OTA), has been used to locate and quantify OT receptors (OTRs) in numerous areas of the rat brain. This ligand has also been applied to locating OTRs in the human brain. The results of the latter studies, however, have been brought into question because of subsequent evidence that (125)I-OTA is much less selective for OTR vs. vasopressin receptors in the primate brain. Previously we used a monoclonal antibody directed toward a region of the human OTR to demonstrate selective immunostaining of cell bodies and fibers in the preoptic-anterior hypothalamic area and ventral septum of a cynomolgus monkey (Boccia et al., 2001). The present study employed the same monoclonal antibody to study the location of OTRs in tissue blocks containing cortical, limbic and brainstem areas dissected from fixed adult, human female brains. OTRs were visualized in discrete cell bodies and/or fibers in the central and basolateral regions of the amygdala, medial preoptic area (MPOA), anterior and ventromedial hypothalamus, olfactory nucleus, vertical limb of the diagonal band, ventrolateral septum, anterior cingulate and hypoglossal and solitary nuclei. OTR staining was not observed in the hippocampus (including CA2 and CA3), parietal cortex, raphe nucleus, nucleus ambiguus or pons. These results suggest that there are some similarities, but also important differences, in the locations of OTRs in human and rodent brains. Immunohistochemistry (IHC) utilizing a monoclonal antibody provides specific localization of OTRs in the human brain and thereby provides opportunity to further study OTR in human development and psychiatric conditions.

Maternal behavior deficits in nulliparous oxytocin knockout mice.

The first observations of postpartum oxytocin knockout (OTKO) mice found no maternal behavior deficits. However, it is unclear how detailed those observations were. In this study, we compared maternal behavior exhibited by OTKO and wild-type (WT) nullipara toward six 2-4-day-old foster pups during test sessions conducted on 3 successive days. Each day, subjects were placed in a clean cage 30 min prior to introduction of pups which were deposited in a clump adjacent to the middle of a long wall of each test cage. Behavior was measured for 3.5 h after which pups and test subjects were returned to their home cages. On test days 1 and 3, a significantly smaller proportion of OTKO females retrieved pups to a corner of their cage. Also, significantly fewer pups were retrieved to corners by OTKO females. In contrast to most WTs, most OTKO females mothered pups in the center of the cage where they were initially deposited. Pup-licking frequencies were significantly lower in OTKO females. Their self-grooming frequencies also trended toward being lower. Latencies to retrieve and lick pups, latencies to and frequencies of still crouching over pups and proportion of time in nest did not differ between groups. Our findings suggest that OT stimulates a significant proportion of pup-licking in nulliparous mice, a situation similar to lactating rat mothers. Our results also indicate that OT may play a role in the motivation to retrieve pups to a more secure location.

Vasopressin interactions with oxytocin in the control of female sexual behavior.

Previous studies have found that central administration of arginine vasopressin and arginine vasopressin receptor V1a antagonists respectively inhibited and stimulated receptivity but did not examine effects on other aspects of female sexual behavior. Central oxytocin facilitates both proceptive and receptive components of sexual behavior and diminishes male-directed agonistic behavior. The present study examined i.c.v.-administered arginine vasopressin and V1a antagonist effects on proceptive, receptive and agonistic behaviors, and interactions with oxytocin. In experiment 1, rats were primed s.c. with 2 microg estradiol benzoate x 2 days and with 500 microg of progesterone on day 3. Arginine vasopressin (0.2, 0.4 microg) or normal saline vehicle was administered 5 h after progesterone treatment and sexual and agonistic behavior measured 30, 60 and 90 min later. Compared with saline, both doses of arginine vasopressin significantly decreased lordosis responses to mounting and hop-dart proceptive behavior and trended toward significantly increasing agonistic behaviors. In experiment 2, oxytocin (2 microg) infusion just after arginine vasopressin (0.4 microg) significantly increased lordoses and decreased agonistic behaviors but did not affect hopping and darting. In experiment 3, conducted in ovariectomized rats primed with estradiol benzoate (1 microg/day s.c. x 2 days), i.c.v. infusion of 0.5 and 1.0 microg of the selective V1a antagonist, d(CH2)5Tyr-(Me)arginine vasopressin on day 3 significantly increased lordoses and trended toward increasing hopping and darting 4 and 6 h after i.c.v. treatment. In experiment 4, 1 microg of the selective oxytocin antagonist, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH2(9)]OVT given 1 h before d(CH2)5Tyr-(Me)arginine vasopressin (1 microg) significantly decreased lordoses. Previous studies indicate that arginine vasopressin contributes to light phase inhibition of female sexual behavior. Our findings suggest that arginine vasopressin may exert this effect through interactions that decrease oxytocin stimulation of sexual behavior and raise the question whether sex steroid conditions that stimulate sexual behavior may suppress central arginine vasopressin and V1a receptor activity.

Potential adverse drug events in an indigent and homeless geriatric population.

OBJECTIVE: To identify potential adverse drug events (ADEs) in a geriatric ambulatory population using the modified Beers criteria. METHODS: This is a cross-sectional study of an indigent and homeless geriatric population served by a network of six primary healthcare clinics with clinical pharmacy services. Medical records of patients > or = 65 years old visiting the clinics between December 1999 and April 2000 were retrospectively reviewed by a clinical pharmacist. Medications meeting the modfied Beers criteria were evaluated for the most common drug classes involved, severity potential, and dose or disease state restrictions. Following the identification of medications meeting Beers criteria, the pharmacist left a written recommendation regarding use of alternative drugs or doses in the medical record. Physician acceptance of pharmacy recommendations was also evaluated. RESULTS: Medical records of 146 patients (71.9% women, average age 72.6 +/- 6.7 y) were reviewed. Overall, 52 patients (35.6%) had 70 medications with the potential for causing an ADE based on the modified Beers criteria The most commonly identified medication classes were narcotic analgesics (20.0%), antihypertensives (20.0%), and antihistamines (14.3%). Fifteen of these medications (21.4%) had a high severity potential. Identified medications met the following modified Beers criteria: 41.4% were inappropriate in a specific disease state, 38.6% were inappropriate for the elderly, 10.0% exceeded maximum dosage guidelines, and 10.0% were inappropriate for both the elerly and the patients disease state. Approximately 60% of pharmacy recommendations were accepted by physicians. CONCLUSIONS: The modified Beers criteria are a useful tool for reviewing medical records to identify potential ADEs in an ambulatory geriatric population.

Brief vs. long maternal separations in infancy: contrasting relationships with adult maternal behavior and lactation levels of aggression and anxiety.

We compared the effects of daily long (3 h), brief (15 min) or no maternal separation (LMS, BMS, NMS) on postnatal days 2-14 on maternal behavior, aggression and anxiety levels during lactation in adulthood. Animals subjected to LMS received less maternal grooming than animals subjected to BMS. Maternal behaviors, including nursing, pup-grooming (PG) frequency and proportion of total grooming (PG+self-grooming) and nest-building during the immediate postpartum period and on postpartum days 2 and 5 were lower in dams with LMS experience compared to dams with BMS experience. LMS dams attacked male rats placed in their home cages less quickly and less often than did BMS or NMS dams. LMS dams also exhibited more anxiety than BMS dams in the elevated plus maze test. Thus, maternal separation during the postnatal period (or associated changes in the amount of maternal grooming received) affected subsequent adult maternal behavior, aggression and anxiety. The mechanism for this remains to be discovered, however, it seems likely to involve alteration of the development of oxytocin receptors in the brain.

Policies on documentation and disciplinary action in hospital pharmacies after a medication error.

Hospital pharmacies were surveyed about policies on medication error documentation and actions taken against pharmacists involved in an error. The survey was mailed to 500 randomly selected hospital pharmacy directors in the United States. Data were collected on the existence of medication error reporting policies, what types of errors were documented and how, and hospital demographics. The response rate was 28%. Virtually all of the hospitals had policies and procedures for medication error reporting. Most commonly, documentation of oral and written reprimand was placed in the personnel file of a pharmacist involved in an error. One sixth of respondents had no policy on documentation or disciplinary action in the event of an error. Approximately one fourth of respondents reported that suspension or termination had been used as a form of disciplinary action; legal action was rarely used. Many respondents said errors that caused harm (42%) or death (40%) to the patient were documented in the personnel file, but 34% of hospitals did not document errors in the personnel file regardless of error type. Nearly three fourths of respondents differentiated between errors caught and not caught before a medication leaves the pharmacy and between errors caught and not caught before administration to the patient. More emphasis is needed on documentation of medication errors in hospital pharmacies.

Allopregnanolone levels and reactivity to mental stress in premenstrual dysphoric disorder.

BACKGROUND: This study was designed to examine basal and stress-induced levels of the neuroactive progesterone metabolite, allopregnanolone, in women with premenstrual dysphoric disorder (PMDD) and healthy control subjects. Also, because evidence suggests that allopregnanolone negatively modulates the hypothalamic-pituitary-adrenal axis, plasma cortisol levels were examined. An additional goal was to investigate the relationship between premenstrual symptom severity and luteal phase allopregnanolone levels. METHODS: Twenty-four women meeting prospective criteria for PMDD were compared with 12 controls during both the follicular and luteal phases of confirmed ovulatory cycles, counterbalancing phase at first testing. Plasma allopregnanolone and cortisol were sampled after an extended baseline period and again 17 min following the onset of mental stress. Owing to low follicular phase allopregnanolone levels, only luteal phase allopregnanolone and cortisol were analyzed. RESULTS: During the luteal phase, PMDD women had significantly greater allopregnanolone levels, coupled with significantly lower cortisol levels, during both baseline and mental stress. Moreover, significantly more controls (83%) showed the expected stress-induced increases in allopregnanolone compared with PMDD women (42%). Premenstrual dysphoric disorder women also exhibited a significantly greater allopregnanolone/progesterone ratio than control subjects, suggesting alterations in the metabolic pathways involved in the conversion of progesterone to allopregnanolone. Finally, PMDD women with greater levels of premenstrual anxiety and irritability had significantly reduced allopregnanolone levels in the luteal phase relative to less symptomatic PMDD women. No relationship between symptom severity and allopregnanolone was observed in controls. CONCLUSIONS: These results suggest dysregulation of allopregnanolone mechanisms in PMDD and that continued investigations into a potential pathophysiologic role of allopregnanolone in PMDD are warranted.

A national survey of U.S. pharmacists in 2000: assessing nonresponse bias of a survey methodology.

The first objective of this study was to assess the existence of nonresponse bias to a national survey of licensed pharmacists conducted in 2000. Three methods were used to assess nonresponse bias. The second objective of the study was to examine reasons why sampled licensed pharmacists did not respond to the national survey of licensed pharmacists. We used data from 2204 respondents to a national survey of pharmacists and from 521 respondents to a survey of nonrespondents to the national survey. We made comparisons between respondents for 5 variables: employment status, gender, age, highest academic degree, and year of initial licensure. Chi-square tests were used to examine differences in the 5 variables between respondents to the first mailing and second mailing of the survey, early and late respondents to the survey, and respondents to the survey and respondents to the nonrespondent survey. There were no significant differences between first mailing and second mailing respondents, but there were differences in each variable except year of licensure between early and late respondents. These differences likely were due to regional bias possibly related to differences in mailing times. There were differences between respondents and nonrespondents in terms of employment status and year of licensure. The main reasons for not responding to the survey were that it was too long or that it was too intrusive. Overall, the survey methodology resulted in a valid sample of licensed pharmacists. Nonresponse bias should be assessed by surveying nonrespondents. Future surveys of pharmacists should consider the length of the survey and the address where it is sent.

ASHP national survey of pharmacy practice in hospital settings: prescribing and transcribing--2001.

Results of the 2001 ASHP national survey of pharmacy practice in hospital settings that pertain to prescribing and transcribing are presented. A stratified random sample of pharmacy directors at 1091 general and children's medical-surgical hospitals in the United States was surveyed by mail. SMG Marketing Group, Inc., supplied data on hospital characteristics; the survey sample was drawn from SMG's hospital database. The response rate was 49.0%. During 2001, nearly all hospitals are estimated to have pharmacy and therapeutics (P&T) committees that meet an average of seven times per year. It is estimated that more than 90% of P&T committees are responsible for formulary development and management, drug policy development, adverse-drug-reaction review, and medication-use evaluation. More than 90% of hospitals use clinical and therapeutic, cost, and pharmacoeconomic information in the formulary management process, while nearly two thirds consider quality-of-life issues. Nearly 70% use clinical practice guidelines in the formulary management process, and 78% have a medication-use evaluation program designed to improve prescribing. Pharmacists in more than 75% of hospitals provide consultations on drug information, dosage adjustments for patients with renal impairment, antimicrobials, and pharmacokinetics. Further, a majority of hospitals ensure accurate transcription of medication orders by clarifying illegible orders before transcription or entry into medication administration records (MARs), using standardized prescriber order forms, requiring prescribers to countersign all oral orders, and reconciling MARs and pharmacy patient profiles at least daily. In 2001, large hospitals are most likely to use prescriber order-entry systems to improve patient safety and are least likely to require the reentry of medication orders into the pharmacy computer system. The 2001 ASHP survey results suggest that pharmacists in hospital settings have positioned themselves well to improve the prescribing and transcribing components of the medication-use process.

ASHP national survey of pharmacy practice in acute care settings: monitoring, patient education, and wellness--2000.

Results of the 2000 ASHP national survey of pharmacy practice in acute care settings that pertain to patient medication monitoring, education, and wellness are presented. Pharmacy directors at 1063 general and children's medical-surgical hospitals in the United States were surveyed by mail. The response rate was 50.2%. Although the respondents indicated that most pharmacists spent less than 20% of their time on medication-monitoring activities, the amount of time devoted to such activities was increasing. Pharmacists were selective about which patients they chose to monitor for medication-related problems. Patients were frequently chosen on the basis of service or medication. Pharmacists used a number of mechanisms to monitor patients for adverse drug events (ADEs). Although internal ADE reporting had generally increased within the preceding three years, 81% of the Institutions had implemented strategies to improve reporting. When ADEs were reported externally (59% of the respondents), FDA was most commonly alerted. About 92% of the respondents indicated that nursing had primary responsibility for counseling patients about medications. Pharmacists were infrequently involved in medication education during the hospital stay; however, 48% of the institutions used some method to identify patients needing counseling by pharmacists. Slightly more than half of the respondents reported having wellness programs. Pharmacists were most commonly involved in disease-based wellness programs. Pharmacists in acute care settings appear to be well positioned to improve the patient-monitoring, education, and wellness components of the medication-use process.

Anti-thrombotic efficacies of enoxaparin, dalteparin, and unfractionated heparin in venous thrombo-embolism.

BACKGROUND: Few data exist by which the anti-thrombotic efficacy of different anticoagulants may be compared. We used a radiolabeled antibody specific for polymerizing fibrin to compare the in vivo anti-thrombotic potencies of different systemic anticoagulants (enoxaparin, dalteparin, and unfractionated heparin). METHODS AND RESULTS: Deep venous thrombi (DVTs) were induced in dogs' femoral veins. The dogs were then treated with one of the following subcutaneous regimens: enoxaparin 100 units/kg (1.0 mg/kg) every 12 hours (n=4), dalteparin 200 units/kg every 24 hours (n=4), or unfractionated heparin 240 units/kg every 8 hours with dose adjustment via aPTT (n=3). 111Indium-labeled anti-fibrin antibodies, specific for propagating thrombi, were given intravenously and nuclear scans of the legs were taken over the following 24 hours. Thrombus propagation was estimated by the ratio of gamma emissions from the legs containing DVTs divided by the emissions from the contralateral "control" legs. DVTs accumulated labeled anti-fibrin antibodies at the same rates in both the enoxaparin group and the dalteparin group (gamma emissions 171+/-6% and 168+/-36% of control by 24 hours, respectively). DVTs in the adjusted dose unfractionated heparin group tended to accumulate antibodies at a slower rate (129+/-19% of control by 24 hours). CONCLUSIONS: Enoxaparin and dalteparin inhibited propagation of pre-formed thrombi to the same degree. Subcutaneous unfractionated heparin, adjusted every 8 hours by aPTT, tended to suppress ongoing thrombosis more than either LMWH.

Pulmonary vascular remodeling distal to pulmonary artery ligation is accompanied by upregulation of endothelin receptors and nitric oxide synthase.

There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8 +/- 2.2 microns vs. 16.8 +/- 0.9 microns in sham group; P < .005) and number of bronchial arteries per bronchiole (3.21 +/- mu 0.26 vs. 1.86 +/- mu 0.21 in sham group; P < .001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These findings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1-mediated vascular remodeling.

Primary care physician incentives in medical group practices.

CONTEXT: Although medical groups are adapting to changes in financing health care, little is known about individual physician incentives in this environment. OBJECTIVES: To describe methods group practices use to compensate primary care physicians in a managed care environment and to examine the association of revenue sources for the group practice from all patients and primary care physician incentives. DESIGN: We surveyed by mail group practice administrators for practices that had at least 200 members continuously enrolled in 1995. SETTING: Group practices that had contractual arrangements with Blue Cross/Blue Shield of Minnesota. PARTICIPANTS: One hundred of 129 group practices returned usable surveys. RESULTS: Most groups had some portion of primary care physicians' compensation at risk, although 17 groups compensated them through fully guaranteed annual salary. Seventy-one groups used productivity, 4 groups used quality of care, 1 group used utilization, and 30 used group financial performance. Factors reported to significantly influence primary care physician compensation included billings or charges, overall group practice performance, and net revenue or profit. Groups that had a higher proportion of income from various types of fee-for-service arrangements used lower proportions of base salary for primary care physician compensation and were more likely to relate physician income to measures of productivity. CONCLUSIONS: Substantial variation exists in the types of primary care physician incentives implemented by medical groups. Base salary, individual productivity, and group financial performance were most frequently used to determine compensation. Physician personal financial risk was higher overall in group practices that derived more revenue from fee-for-service contracts.

Endothelin mediates pulmonary vascular remodelling in a canine model of chronic embolic pulmonary hypertension.

It is well known that endothelin (ET)-1 mediates vascular remodelling in various kinds of clinical and experimental pulmonary hypertension. The aim of this study was to investigate whether ET-1 is associated with the development of pulmonary vascular remodelling in a canine model of chronic embolic pulmonary hypertension. Pulmonary hypertension was induced in 10 mongrel dogs by repeated embolization with ceramic beads. In five of the dogs, bosentan, a nonselective ET receptor antagonist, was administered throughout the study. Haemodynamic measurements and plasma ET-1 assays were performed every 2 months. Eight months after initial embolization, computer-assisted morphometry and immunohistochemistry were performed on the lung tissue including that from three control dogs. Pulmonary arterial pressure and pulmonary vascular resistance were increased in all embolized dogs, compared to baseline. In nontreated embolized dogs, plasma ET-1 concentration and pulmonary arterial wall thickness were increased compared to control animals, and ET-1 immunoreactivity was detected in thickened pulmonary arteries. In bosentan treated dogs, pulmonary arterial walls were not significantly thickened. Pulmonary vascular remodelling, associated with elevated plasma endothelin-1 levels and positive endothelin-1 immunoreactivity in lung tissue is attenuated by the endothelin receptor antagonist, bosentan. These findings suggest that endothelin mediates pulmonary vascular remodelling in a canine model of chronic embolic pulmonary hypertension.

Strychnine alkaloid and avian reproduction: effects occur at lower dietary concentrations with mallard ducks than with bobwhite quail.

Separate subchronic reproductive toxicity studies were conducted using mallard ducks (Anas platyrhynchos) and northern bobwhite quail (Colinus virginianus). Three groups (32/group; 16 male-female pairs) of 17-week-old ducks (F0 generation) were fed Purina Game Bird Breeder Layena diets containing mean (+/-SD) 33.2 (+/-2.7), 68.9 (+/-1.8), and 140.9 (+/-5.1) microg/g strychnine for 20 weeks, with some pairs in each group fed control diet during a subsequent 3-week recovery period. Three groups (32/group; 16 male-female pairs) of 19-week-old quail (F0 generation) were fed similar diets containing mean (+/-SD) 279.2 (+/-10.1), 557.4 (+/-43.5), and 1,113.6 (+/-46.6) microg/g strychnine for 22 weeks without a recovery period. Separate groups of ducks and quail (32/group; 16 male-female pairs) were also fed control diets (0.0 microg/g strychnine) in each study. There were 16 weekly collections of eggs for the mallard study (13 for the diet-exposure period and 3 for the recovery period), and 11 collections for the quail study. Eggs laid during the last 13 and 10 weeks of the diet-exposure periods for ducks (plus 3 weeks of the recovery period) and quail, respectively, were incubated. Each hatch of F1 generation ducklings and chicks was observed for 14 days. Key results were: (1) the no observed adverse effect levels (NOAELs) for F0 ducks and quail were 33.2 and 1,113.6 microg/g strychnine, respectively--quail showed no reproductive effects at the current doses; (2) decreased egg production and hatching success occurred for mallard hens fed mean 140.9 microg/g strychnine diets; and (3) "normal-hatching" ducklings from eggs of F0 mallards fed mean 140.9 microg/g strychnine diets suffered greater mortality than ducklings from the other diet groups. Possible mechanisms of strychnine action on avian reproduction are discussed.

Postpartum mood and anxiety disorders: a guide for the nonpsychiatric clinician with an aside on thyroid associations with postpartum mood.

This article summarizes the considerable progress that has been made in recent years in our understanding of the risks of pregnancy-associated mood and anxiety disorders, effective somatic and nonsomatic treatments for those disorders, and the risks and benefits of psychotropic medications during pregnancy and breast-feeding. Ten to 15% of women develop syndromal depressions within the first 2 to 3 months postpartum, making this the most common serious medical complication of the puerperium. Prior history of depressive disorders, lack of social support, and stressful life events all increase the risk of postpartum depression. Psychotic mood disorders (commonly called postpartum psychosis) occur after approximately 1 to 2 deliveries per 1000. To ensure safety, women who develop postpartum psychotic symptoms should be hospitalized. History of bipolar illness increases the risk of postpartum psychosis to as much as 25%. Prior episodes of postpartum psychosis further increase the risk to 50%-75%. The risk of first onset or exacerbation of panic disorder or obsessive-compulsive disorder appears to increase during pregnancy and the puerperium. Postpartum mood and anxiety disorders are highly responsive to psychotherapy and in more serious cases, to medication. Fortunately, several classes of psychotropic medications, especially tricyclic and selective serotonin reuptake inhibitor antidepressants, appear to be reasonable safe during pregnancy and breast-feeding. Electroconvulsive therapy is the most effective treatment for very severe postpartum mood disorders, including postpartum psychosis. Preliminary results are also presented that suggest that lower range total and free thyroxine concentrations during late pregnancy are related to postpartum depressive symptoms.

Radioligand assays for oestradiol and progesterone conjugated to protein reveal evidence for a common membrane binding site in the medial preoptic area-anterior hypothalamus and differential modulation by cholera toxin and GTPgammaS.

In this study membrane oestradiol (E) binding sites in the medial preoptic area-anterior hypothalamus (MPOA-AH) of ovariectomized (OVX) rats were characterized using standard radioligand binding techniques employing E conjugated to bovine serum albumin (BSA) at position 6 and radiolabeled with 125I (E-6-[125I-BSA]). In previous studies binding of a radioactive conjugate of progesterone (P) and BSA (P-3-[125I-BSA]) was examined using the same membrane preparation. E-6-[125I-BSA] binding was linear across a tissue concentration range of 0.005-0.02 mg protein/0.1 ml of membrane suspension. An association T1/2 of 9.5 min and a dissociation T1/2 of 52.1 min for E-6-[125I-BSA] were derived from kinetic experiments. Competition binding experiments revealed high (Ki=0.63+/-(0.50 nM) and low (Ki=161.5(96.5 nM) affinity binding sites for E-6-[125I-BSA], demonstrating different binding parameters than shown in our previous work for P-3-[125I-BSA] binding. Further studies on MPOA-AH membranes treated with cholera toxin (CTX) and GTPgammaS suggested that E-6-BSA binding sites are associated with G proteins. E-6-[125I-BSA] binding demonstrated both high-and low-affinity sites. GTPgammaS added to the assay reduced both E-6-[125I-BSA] and P-3-[125I-BSA] binding suggesting that G proteins are associated with both binding sites. Extensive analysis of both E-6-[125I-BSA] and P-3-[125I-BSA] binding sites demonstrated a reciprocal relationship such that high-affinity E-6-[125I-BSA] binding sites exhibit low affinity for P-3-[125I-BSA] and low-affinity E-6-[125I-BSA] binding sites exhibit high affinity for P-3-[125I-BSA]. Preincubating membranes with CTX or GTPgammaS reduced high-affinity E-6-[125I-BSA] binding and enhanced high-affinity P-3-[125I-BSA] binding. These results suggest that, in the MPOA-AH, membrane steroid binding sites exist in two interconvertible conformations that preferentially bind either E-6-BSA or P-3-BSA, depending on their association with a G protein. Additional studies with free steroids revealed that: (1) oestrogens (17beta-oestradiol, diethylstilbestrol) as well as synthetic oestrogen antagonists tamoxifen and ICI 182 780 displaced P-3-[125I-BSA] further suggesting a relationship between membrane binding sites for E and P-3-[125I-BSA] binding sites; and (2) treatment of OVX rats with E decreased displacement by P-3-BSA and increased displacement by ICI 182,780 and tamoxifen suggesting these antagonists affect membrane P-3-[125I-BSA] binding sites after in-vivo E treatment. The membrane binding sites for E and P demonstrate interrelationships not demonstrated by their nuclear receptors.