Osteoprotegerin mediates tumor-promoting effects of Interleukin-1beta in breast cancer cells.

BACKGROUND: It is widely recognized that inflammation promotes breast cancer invasion and metastasis. Given the complex nature of the breast tumor inflammatory microenvironment, much remains to be understood of the molecular mechanisms that govern these effects. We have previously shown that osteoprotegerin knockdown in breast cancer cells resulted in reduced invasion and metastasis. Here we present novel insight into the role of osteoprotegerin in inflammation-driven tumor progression in breast cancer by investigating the link between osteoprotegerin, macrophages and the potent pro-inflammatory cytokine Interleukin-1beta. METHODS: We used human breast cancer cell lines to investigate the effects of Interleukin-1beta treatment on osteoprotegerin secretion as measured by ELISA. We analyzed public datasets containing human breast cancer genome-wide mRNA expression data to reveal a significant and positive correlation between osteoprotegerin mRNA expression and the mRNA expression of Interleukin-1beta and of monocyte chemoattractant protein CC-chemokine ligand 2. Osteoprotegerin, Interleukin-1beta and CC-chemokine ligand 2 mRNA levels were also examined by qPCR on cDNA from normal and cancerous human breast tissue. We determined the effect of Interleukin-1beta-producing macrophages on osteoprotegerin expression by co-culturing breast cancer cells and differentiated THP-1 macrophages. Immunohistochemistry was performed on human breast tumor tissue microarrays to assess macrophage infiltration and osteoprotegerin expression. To demonstrate that osteoprotegerin mediated functional effects of Interleukin-1beta we performed cell invasion studies with control and OPG siRNA knockdown on Interleukin-1beta-treated breast cancer cells. RESULTS: We report that Interleukin-1beta induces osteoprotegerin secretion, independent of breast cancer subtype and basal osteoprotegerin levels. Co-culture of breast cancer cells with Interleukin-1beta-secreting macrophages resulted in a similar increase in osteoprotegerin secretion in breast cancer cells as Interleukin-1beta treatment. Macrophage infiltration correlates with osteoprotegerin secretion in human breast tumor tissue samples. We show that osteoprotegerin secretion is regulated by Interleukin-1beta in a p38- and p42/44-dependent manner. We also demonstrate that osteoprotegerin knockdown represses Interleukin-1beta expression, Interleukin-1beta-mediated breast cancer cell invasion and MMP3 expression. CONCLUSIONS: These data indicate a novel role for osteoprotegerin as a mediator of inflammation- promoted breast cancer progression.

Osteoprotegerin in breast cancer: beyond bone remodeling.

Osteoprotegerin (OPG) is a secreted protein and member of the Tumor Necrosis Factor (TNF) Receptor superfamily. OPG has been well characterized as a regulator of bone metabolism which acts by blocking osteoclast maturation and preventing bone breakdown. Given this role, early studies on OPG in breast cancer focused on the administration of OPG in order to prevent the osteolysis observed with bone metastases. However OPG is also produced by the breast tumor cells themselves. Research focusing on OPG produced by breast tumor cells has revealed actions of OPG which promote tumor progression. In vitro studies into the role of OPG produced by breast tumor cells have demonstrated that OPG can block TNF-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Furthermore, in vivo studies show that OPG expression by breast tumors can promote tumor growth and metastasis. In addition it has been shown that OPG stimulates endothelial cell survival and tube formation thus it may indirectly promote breast tumor progression through impacting angiogenesis. This article will present a summary of the data concerning the tumor-promoting effects of OPG in breast cancer.

"It Made Me Think Like a Nurse": Virtual Simulations With Interpreters.

BACKGROUND: Simulations are a beneficial educational exercise for nursing students and students studying interpreting. Leveraging videoconferencing software is one way to incorporate virtual experiential learning simulations into curricula. METHOD: Virtual simulations of patient education interactions were conducted with nursing and standardized patients who are deaf. The virtual consultations were mediated by interpreting students. Following the simulations, data were collected from students about their experiences. Thematic analysis was used to identify overarching patterns. RESULTS: Results indicated students reported myriad learning outcomes and areas of growth, as well as several logistical challenges. Students from both disciplines reported new learning experiences and development of practical skills, as well as critical thinking abilities. CONCLUSION: Virtual simulations of health care appointments between nursing students and deaf standardized patients are a worthwhile educational endeavor and lead to positive learning outcomes for all of the students involved. [J Nurs Educ. 2022;61(6):334-337.].

Targeted therapy for breast cancer: An overview of drug classes and outcomes.

The last 25 years have seen significant growth in new therapeutic options for breast cancer, termed targeted therapies based on their ability to block specific pathways known to drive breast tumor growth and survival. Introduction of these drugs has been made possible through advances in the understanding of breast cancer biology. While the promise of targeted therapy for breast cancer has been clear for some time, the experience of the clinical use of multiple drugs and drug classes allows us to now present a summary and perspective as to the success and impact of this endeavor. Here we will review breast cancer targeted therapeutics in clinical use. We will provide the rationale for their indications and summarize clinical data in patients with different breast cancer subtypes, their impact on breast cancer progression and survival and their major adverse effects. The focus of this review will be on the development that has occurred within classes of targeted therapies and subsequent impact on breast cancer patient outcomes. We will conclude with a perspective on the role of targeted therapy in breast cancer treatment and highlight future areas of development.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model.

INTRODUCTION: Metastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs. METHODS: To investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process. RESULTS: We found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases. CONCLUSIONS: Our results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection.

We investigated whether nuclear factor kappa B (NF-kappaB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re-modeling. We generated a doxycycline inducible transgenic mouse model to activate NF-kappaB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF-kappaB signaling cascade. We found that activation of NF-kappaB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase-3, an indicator of apoptosis. We also found that activation of NF-kappaB during lactation with no additional involution signals had a similar effect. The observation that NF-kappaB is a key regulator of milk production led us to investigate the role of NF-kappaB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF-kappaB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF-kappaB in mammary epithelium. Together, our data reveal that activation of NF-kappaB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF-kappaB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact.

Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis.

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-kappaB, a central effector of inflammatory responses, is frequently activated in non-small-cell lung cancer, but its role in tumor promotion has not been studied. Several lines of evidence indicate that ethyl carbamate (urethane)-induced lung tumor formation, a prototypical mouse model of multistage lung carcinogenesis, is potentiated by inflammation. We found that mouse strains susceptible to lung tumor formation (FVB, BALB/c) exhibited early NF-kappaB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-kappaB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-kappaB activation in airway epithelium, as well as type II alveolar epithelial cells and macrophages. Using an inducible transgenic mouse model (FVB strain) to express a dominant inhibitor of NF-kappaB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-kappaB inhibition resulted in increased apoptosis of airway epithelial cells at 2 weeks after urethane treatment in association with a marked reduction of Bcl-2 expression. These studies indicate that NF-kappaB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-kappaB pathway as a potential target for chemoprevention of lung cancer.

Osteoprotegerin: Relationship to Breast Cancer Risk and Prognosis.

Osteoprotegerin (OPG) is a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily (TNFRSF11B), that was first characterized and named for its protective role in bone remodeling. In this context, OPG binds to another TNF superfamily member Receptor Activator of NF-kappaB Ligand (RANKL; TNFSF11) and blocks interaction with RANK (TNFRSF11A), preventing RANKL/RANK stimulation of osteoclast maturation, and bone breakdown. Further studies revealed that OPG protein is also expressed by tumor cells and led to investigation of the role of OPG in tumor biology. An increasing body of data has demonstrated that OPG modulates breast tumor behavior. Initially, research was focused on OPG in the bone microenvironment as a potential inhibitor of RANKL-driven osteolysis. More recently, attention has shifted to include OPG expression and interactions in the primary breast tumor independent of RANKL. In the primary tumor, OPG may interact with another TNF superfamily member, TNF-Related Apoptosis Inducing Ligand (TRAIL; TNFSF10) to prevent apoptosis induction. Additional interest in OPG in breast cancer has been stimulated by the tumor-promoting role of its binding partner RANKL in association with BRCA1 gene mutations. We and others have previously summarized the functional studies on OPG and breast cancer (1, 2). After basic research studies on the in vitro role for OPG (and RANKL) in breast cancer, the field now expands to assess the in vivo role for OPG by examining the correlation between OPG expression and breast cancer risk or patient prognosis. However, the data reported so far is conflicting, since OPG expression appears linked to both good and poor patient survival. In the current review we will summarize these studies. Our goal is to provide stimulus for further research to bridge the basic research findings and clinical data regarding OPG in breast cancer.

Breast Adipocyte Co-culture Increases the Expression of Pro-angiogenic Factors in Macrophages.

Obese individuals with breast cancer have a poorer prognosis and higher risk of metastatic disease vs. non-obese patients. Adipose tissue in obese individuals is characterized by an enhanced macrophage infiltration, creating a microenvironment that favors tumor progression. Here, we demonstrate a role for adipocyte-macrophage interactions in the regulation of angiogenesis. Co-culture of THP-1 macrophages with human breast adipocytes led to increased expression of the pro-angiogenic growth factor, vascular endothelial growth factor A (VEGFA). Several adipocyte-derived proteins including leptin, insulin, IL-6, and TNF-α were each capable of increasing VEGFA expression in THP-1 macrophages, identifying these as possible mediators of the changes that were observed with co-culture. Furthermore, analysis of THP-1 culture media by antibody array revealed that THP-1 secrete several other pro-angiogenic signals in response to adipocyte co-culture, including interleukin 8 (IL-8), matrix metalloproteinase 9 (MMP9), pentraxin 3 (PTX3), and serpin E1 (plasminogen activator inhibitor 1, PAI1) after co-culture with human adipocytes. We used an in vitro endothelial tube formation assay with human vascular endothelial cells to evaluate the effects of THP-1 culture media on angiogenesis. Here, culture media from THP-1 cells previously exposed to human adipocytes stimulated endothelial tube formation more significantly than THP-1 cells cultured alone. In summary, we find that adipocyte co-culture stimulates the expression of pro-angiogenic mediators in macrophages and has pro-angiogenic effects in vitro, thus representing a possible mechanism for the enhanced risk of breast cancer progression in obese individuals.

Can nursing students' confidence levels increase with repeated simulation activities?

In 2014, nursing faculty conducted a study with undergraduate nursing students on their satisfaction, confidence, and educational practice levels, as it related to simulation activities throughout the curriculum. The study was a voluntary survey conducted on junior and senior year nursing students. It consisted of 30 items based on the Student Satisfaction and Self-Confidence in Learning and the Educational Practices Questionnaire (Jeffries, 2012). Mean averages were obtained for each of the 30 items from both groups and were compared using T scores for unpaired means. The results showed that 8 of the items had a 95% confidence level and when combined the items were significant for p <.001. The items identified were those related to self-confidence and active learning. Based on these findings, it can be assumed that repeated simulation experiences can lead to an increase in student confidence and active learning.

Interleukin-5 facilitates lung metastasis by modulating the immune microenvironment.

Although the lung is the most common metastatic site for cancer cells, biologic mechanisms regulating lung metastasis are not fully understood. Using heterotopic and intravenous injection models of lung metastasis in mice, we found that IL5, a cytokine involved in allergic and infectious diseases, facilitates metastatic colonization through recruitment of sentinel eosinophils and regulation of other inflammatory/immune cells in the microenvironment of the distal lung. Genetic IL5 deficiency offered marked protection of the lungs from metastasis of different types of tumor cells, including lung cancer, melanoma, and colon cancer. IL5 neutralization protected subjects from metastasis, whereas IL5 reconstitution or adoptive transfer of eosinophils into IL5-deficient mice exerted prometastatic effects. However, IL5 deficiency did not affect the growth of the primary tumor or the size of metastatic lesions. Mechanistic investigations revealed that eosinophils produce CCL22, which recruits regulatory T cells to the lungs. During early stages of metastasis, Treg created a protumorigenic microenvironment, potentially by suppressing IFNγ-producing natural killer cells and M1-polarized macrophages. Together, our results establish a network of allergic inflammatory circuitry that can be co-opted by metastatic cancer cells to facilitate lung colonization, suggesting interventions to target this pathway may offer therapeutic benefits to prevent or treat lung metastasis.

Osteoprotegerin expression in triple-negative breast cancer cells promotes metastasis.

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that has been well characterized as a negative regulator of bone remodeling. OPG is also expressed in human breast cancer tissues and cell lines. In vitro studies suggest that OPG exerts tumor-promoting effects by binding to TNF-related apoptosis inducing ligand (TRAIL), thereby preventing induction of apoptosis. However, the in vivo effect of OPG expression by primary breast tumors has not been characterized. We knocked down OPG expression in MDA-MB-231 and MDA-MB-436 human breast cancer cells using shRNA and siRNA to investigate impact on metastasis in the chick embryo model. We observed a reduction in metastasis with OPG knockdown cells. We found that lowering OPG expression did not alter sensitivity to TRAIL-induced apoptosis; however, the OPG knockdown cells had a reduced level of invasion. In association with this we observed reduced expression of the proteases Cathepsin D and Matrix Metalloproteinase-2 upon OPG knockdown, indicating that OPG may promote metastasis via modulation of protease expression and invasion. We conclude that OPG has a metastasis-promoting effect in breast cancer cells.

Silent information regulator (Sir)T1 inhibits NF-κB signaling to maintain normal skeletal remodeling.

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF-κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF-κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast- or osteoblast-specific SirT1 deletion using the Sirt(flox/flox) mice crossed to lysozyme M-cre and the 2.3 kb col1a1-cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF-κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF-κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF-κB. In further support of the critical role of NF-κB signaling in bone remodeling, elevated NF-κB activity in IκBα(+/-) mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell-autonomous manner in both osteoblasts and osteoclasts, through control of NF-κB and bone cell differentiation.

Intraductal injection of LPS as a mouse model of mastitis: signaling visualized via an NF-κB reporter transgenic.

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-κB) signaling, as visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse. Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-κB luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-κB signaling within the LPS-injected gland. These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Nursing and en route care: history in time of war.

The mission of the en route caregiver is to provide critical care in military helicopters for wounded Warriors. This care minimizes the effects of the wounds and injuries, and improves morbidity and mortality. This article will focus on the history of Army Nursing en route care. From World War II through Vietnam, and continuing through the War on Terrorism in Iraq and Afghanistan, Army nurses served in providing en route care in military airplanes and helicopters for patients being transported to higher echelons of care. From aid stations on the battlefield to forward surgical teams which provide life, limb, and eyesight saving care, to the next higher level of care in combat support hospitals, these missions require specialized nursing skills to safely care for the high acuity patients. Before the en route care concept existed, there was not a program to train nurses in these critical skills. There was also a void of information about patient outcomes associated with the nursing assessment and care provided during helicopter medical evacuation (MEDEVAC) of such unstable patients, and the consequent impact on the patient's condition after transport. The role of critical care nurses has proven to be essential and irreplaceable in providing full-spectrum care to casualties of war, in particular, the postsurgical patients transferred from one surgical facility to another in theatre. However, we have only recently developed the concepts over the required skill set, training, equipment, functionality, evidenced-based care, and sustainability of nursing in the en route care role. Much of the work to quantify and qualify nursing care has been done by individuals and individual units whose lessons-learned have only recently been captured.

The nuclear factor-kappaB pathway controls the progression of prostate cancer to androgen-independent growth.

Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an "androgen-independent" stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappaB (NF-kappaB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-kappaB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-kappaB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-kappaB signaling in vivo by the absence of the IkappaBalpha inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-kappaB pathway was activated in the ARR(2)PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IkappaBalpha(+/-) haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-kappaB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-kappaB pathway may be a potential target for therapy against androgen-independent prostate cancer.

A transgenic model reveals important roles for the NF-kappa B alternative pathway (p100/p52) in mammary development and links to tumorigenesis.

A regulated pattern of nuclear factor kappaB (NF-kappaB) activation is essential for normal development of the mammary gland. An increase in NF-kappaB activity has been implicated in breast cancer. We have generated a novel transgenic mouse model to investigate the role of the alternative NF-kappaB pathway in ductal development and identify possible mediators of tumorigenesis downstream of p100/p52. By overexpressing the NF-kappaB p100/p52 subunit in mammary epithelium using the beta-lactoglobulin milk protein promoter, we found that transgene expression resulted in increased overall NF-kappaB activity during late pregnancy. During pregnancy, p100/p52 expression resulted in delayed ductal development with impaired secondary branching and increased levels of Cyclin D1, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and cyclo-oxygenase-2 (COX-2) in the mammary gland. After multiple pregnancies the p100 transgenics exhibited a ductal thickening accompanied by small hyperplastic foci. In tumors from mice expressing the polyoma middle T oncoprotein (PyVT) in the mammary gland, increased levels of p100/p52 were present at the time of tumor development. These results show that increased p100/p52 disrupts normal ductal development and provides insight into the mechanism by which this may contribute to human breast cancer.

Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice: a pro-inflammatory role for eNOS-derived no in vivo.

The expression of inducible nitric-oxide synthase (iNOS) and subsequent "high-output" nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.

Tracazolate reveals a novel type of allosteric interaction with recombinant gamma-aminobutyric acid(A) receptors.

Tracazolate, a pyrazolopyridine, is an anxiolytic known to interact with gamma-aminobutyric acid (GABA)(A) receptors, adenosine receptors, and phosphodiesterases. Its anxiolytic effect is thought to be via its interaction with GABA(A) receptors. We now report the first detailed pharmacological study examining the effects of tracazolate on a range of recombinant GABA(A) receptors expressed in Xenopus laevis oocytes. Replacement of the gamma2s subunit within the alpha1beta3gamma2s receptor with the epsilon subunit caused a dramatic change in the functional response to tracazolate from potentiation to inhibition. The gamma2s subunit was not critical for potentiation because alpha1beta3 receptors were also potentiated by tracazolate. gamma2/epsilon chimeras revealed a critical N-terminal domain between amino acids 206 and 230 of gamma2, governing the nature of this response. Replacement of the beta3 subunit with the beta1 subunit within alpha1beta3gamma2s and alpha1beta3epsilon receptors also revealed selectivity of tracazolate for beta3-containing receptors, determined by asparagine at position 265 within transmembrane 2. Replacement of gamma2s with gamma1 or gamma3 revealed a profile intermediate to that of alpha1beta1epsilon and alpha1beta1gamma2s. alpha1beta1delta receptors were also potentiated by tracazolate; however, the maximum potentiation of the EC(20) was much greater than on alpha1beta1gamma2. Concentration-response curves to GABA in the presence of tracazolate for alpha1beta1epsilon and alpha1beta1gamma2s revealed a concentration-related decrease in maximum current amplitude, but a leftward shift in the EC(50) only on alpha1beta1gamma2. Like alpha1beta1gamma2s, GABA concentration-response curves on alpha1beta1delta receptors were shifted to the left with increased maximum responses. Tracazolate has a unique pharmacological profile on recombinant GABA(A) receptors: its potency (EC(50)) is influenced by the nature of the beta subunit; but more importantly, its intrinsic efficacy, potentiation, or inhibition is determined by the nature of the third subunit (gamma1-3, delta, or epsilon) within the receptor complex.