Previously we demonstrated that the heterotrimeric G protein, G alpha i-2, inhibits cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl-) channels in human airway epithelial cells (E. M. Schwiebert, F. Gesek, L. Ercolani, C. Wjasow, D. C. Gruenert, and B. A. Stanton. Am. J. Physiol. 267 (Cell Physiol. 36): C272-C281, 1994, and E. M. Schwiebert, N. L. Kizer, D. C. Gruenert, and B. A. Stanton. Proc. Natl. Acad. Sci. USA 89: 10623-10627, 1992). The goal of the present study was to determine if G proteins also regulate outwardly rectifying Cl- channels (ORCC), a distinct class of Cl- channels regulated defectively by protein kinase A (PKA) in cystic fibrosis (CF). To this end, we used the patch-clamp technique to study ORCC in a normal human airway epithelial cell line (9HTEo-) that expresses CFTR and ORCC. Stimulation of G proteins with GTP and GTP gamma S decreased the single-channel open probability (Po) of ORCC, whereas inhibition of G proteins by GDP beta S increased the Po. Moreover, pertussis toxin (PTX), an uncoupler of Gi and G(o) subclasses of heterotrimeric G proteins, also increased the Po. Purified G alpha i-2 decreased the Po. In contrast, other PTX-sensitive G proteins, G alpha i-1, G alpha i-3, and G alpha o, had no effect on Po. We propose that G alpha i-2 couples to a receptor whose agonist negatively regulates ORCC in human airway epithelial cells.

P2 receptors are potent modulators of key solute and water transport mechanisms in the renal collecting duct (CD) [1]. Controversy exists over the P2 receptor subtype(s) responsible; most studies focus on metabotropic P2Y receptors and arguably ionotropic P2X receptors are largely overlooked. Using an M1 cell line derived from mouse CD and the excised outside‐out configuration of the patch‐clamp technique we have sought functional evidence for extracellular ATP‐gated P2X receptor ion channels. External bath solution contained 145 mM NaCl and the pipette solution contained 145 mM K ⁺ (70 mM Cl ⁻ ). In outside‐out patches, application of external ATP (100 μM) transiently activated flickery single channel currents (t _act ~3s, t _des ~20s). At −100 mV, the mean inward single channel current amplitude (i), open probability (Po) and chord conductance of channels were 0.71±0.12 pA, 0.66±0.12, and 7.7±0.8 pS (n=5), respectively. We demonstrate that ATP directly activates receptors with singlechannel properties similar to those of P2X receptors. Consequently P2X receptors may regulate CD solute and water transport. Research supported by the Wellcome Trust, BBSRC and DiscoveryBiomed.

Ovarian cancer (OC) is the deadliest gynecological disease in women, with high-grade serous ovarian cancer (HGSC) being its most common and lethal subtype. This disease accounts for 75% of OC cases and has a five-year survival rate of only 32%, mainly due to diagnoses at an advanced stage. Inheriting a pathogenic BRCA1 or 2 mutation significantly increases the risk of developing HGSC. However, the early molecular processes that lead to this deadly subtype remain poorly understood. The ovarian hormone progesterone (P4) has been shown to induce metastatic HGSC in Dicer1-Pten double-knockout (DKO) mice, an animal model that develops this specific type of OC with molecular, histological, and clinical features similar to those of BRCA1/2 mutation carriers. To explore P4-induced metabolic changes before and after the onset of HGSC, we analyzed serum samples from DKO mice treated with P4 or mifepristone, an inhibitor of P4 signaling, at premalignant and early tumor stages. These samples underwent both targeted and non-targeted metabolomic analysis using ultra-high performance liquid chromatography-mass spectrometry. The non-targeted data revealed significant trends among various phospholipid classes, phosphatidylcholines, sphingomyelins, and triacylglycerols, in early-stage HGSC. Additionally, two metabolites previously linked to OC, lysophosphatidylethanolamine (18:1) and tetrahydrocortisone, were significantly elevated at the premalignant stage of HGSC development. Conversely, at this same stage, the targeted dataset showed notable increases in estrogens and glucocorticoids, while higher corticosterone levels were detected as HGSC began to develop. Overall, this study highlights the disruption of specific metabolites, phospholipid classes, and steroid hormones, in relation to HGSC tumor development under P4 treatment, suggesting their potential roles in OC development.

Background: Ovarian cancer (OC) remains one of the most lethal cancers among women due to most cases going undiagnosed until later stages. The early detection and treatment of this malignancy provides the best prognosis, but the lack of an accurate and sensitive screening tool combined with ambiguous symptoms hinders these diagnoses. In contrast, screening for cervical cancer via Papanicolaou (Pap) tests is a widespread practice that greatly reduces the cancer’s mortality rates. Interestingly, previous studies show evidence of OC cells in Pap tests, suggesting that proteins, and potentially lipids, shed from ovarian tumors end up in the cervix. The goal of this study is to evaluate the practicality of using Pap tests as biospecimens for OC-screening-related metabolomics. Methods: To evaluate the effectiveness of using residual Pap test samples as biospecimens for potential metabolomics work, 29 Pap test samples, collected from women over the age of 50 with normal cytology and no visible blood contamination, were first obtained from the University of Minnesota, with IRB approval. These samples were centrifuged to recover the cell pellets from the supernatants. The cell pellets underwent a biphasic extraction, followed by an RP-LC-MS analysis, while the supernatants underwent two separate extractions and analyses, including RP-LC-MS and HILIC-LC-MS. Non-targeted features were detected in the range of 220–1000 m/z to determine the sensitivity and scope of the various extraction and analytical workflows, as well as evaluating residual Pap test samples as viable metabolomics biospecimens. Results: The biphasic extraction and subsequent RP-LC-MS analysis of the isolated cell pellets from all 29 samples yielded informative, exploratory data, highlighting the potential of using residual Pap test samples as biospecimens for metabolomics, specifically lipidomics, studies. Each sample was analyzed in both the positive and negative ion mode, yielding the detection of 7318 in the positive ion mode and 3733 in the negative ion mode. Using multiple reference libraries, 22.85% and 36.19% of these features were annotated in the positive and negative ion mode, respectively. Among these detected features, 453 unique lipids, representative of 20 different lipid subclasses, were annotated in all 29 samples. Of the various lipid subclasses represented from the detected lipids, ceramides, triacylglycerols, hexosylceramides, and phosphatidylcholines contributed to over half (53.3%) of the detected lipids at 16.2%, 13.0%, 12.8%, and 11.3%, respectively. Conclusions: The detection of these 453 common lipids across all patients establishes a relative lipidome baseline for women over the age of 50 with normal cervical cytology. This exploratory study is the first investigation to utilize residual Pap test samples as biospecimens in a metabolomics/lipidomics workflow.

Understanding the biological mechanisms behind fracture nonunions and their treatments remains incomplete. Since these mechanisms are linked to phenotypic expression, studying the longitudinal metabolic profile of bone regeneration has emerged as a potential way to better understand healing progress and treatment effectiveness. In this study, we tracked the serum metabolome of rats with critically-sized femoral defects treated with either a control, a minimum effective dose, or a supraphysiologic dose of rhBMP-2 over time. We observed distinct changes in the metabolome across control, impaired healing, and rhBMP-2-treated animals. Early in the process, we saw increased levels of metabolites that support angiogenesis in the rhBMP-2 groups. Supraphysiologic doses of rhBMP-2 significantly affected metabolite expression, especially in pathways related to angiogenesis, fatty acid metabolism, and ATP production. These longitudinal trends and individual metabolite changes provide a deeper understanding of rhBMP-2’s mechanisms, improve our knowledge of its biological effects—guiding appropriate dosing—and offer a metabolic profile that may indicate healing impairment.