Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels.

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

A comparison of iopromide and iopamidol, two acidoCEST MRI contrast media that measure tumor extracellular pH.

Acidosis within tumor and kidney tissues has previously been quantitatively measured using a molecular imaging technique known as acidoCEST MRI. The previous studies used iopromide and iopamidol, two iodinated contrast agents that are approved for clinical CT diagnoses and have been repurposed for acidoCEST MRI studies. We aimed to compare the performance of the two agents for measuring pH by optimizing image acquisition conditions, correlating pH with a ratio of CEST effects from an agent, and evaluating the effects of concentration, endogenous T1 relaxation time and temperature on the pH-CEST ratio correlation for each agent. These results showed that the two agents had similar performance characteristics, although iopromide produced a pH measurement with a higher dynamic range while iopamidol produced a more precise pH measurement. We then compared the performance of the two agents to measure in vivo extracellular pH (pHe) within xenograft tumor models of Raji lymphoma and MCF-7 breast cancer. Our results showed that the pHe values measured with each agent were not significantly different. Also, iopromide consistently measured a greater region of the tumor relative to iopamidol in both tumor models. Therefore, an iodinated contrast agent for acidoCEST MRI should be selected based on the measurement properties needed for a specific biomedical study and the pharmacokinetic properties of a specific tumor model.

Evaluating pH in the Extracellular Tumor Microenvironment Using CEST MRI and Other Imaging Methods.

Tumor acidosis is a consequence of altered metabolism, which can lead to chemoresistance and can be a target of alkalinizing therapies. Noninvasive measurements of the extracellular pH (pHe) of the tumor microenvironment can improve diagnoses and treatment decisions. A variety of noninvasive imaging methods have been developed for measuring tumor pHe. This review provides a detailed description of the advantages and limitations of each method, providing many examples from previous research reports. A substantial emphasis is placed on methods that use MR spectroscopy and MR imaging, including recently developed methods that use chemical exchange saturation transfer MRI that combines some advantages of MR spectroscopy and imaging. Together, this review provides a comprehensive overview of methods for measuring tumor pHe, which may facilitate additional creative approaches in this research field.

Evaluations of Tumor Acidosis Within In Vivo Tumor Models Using Parametric Maps Generated with Acido CEST MRI.

PURPOSE: We aimed to develop pixelwise maps of tumor acidosis to aid in evaluating extracellular tumor pH (pHe) in cancer biology. PROCEDURES: MCF-7 and MDA-MB-231 mouse models were imaged during a longitudinal study. AcidoCEST MRI and a series of image processing methods were used to produce parametric maps of tumor pHe, and tumor pHe was also measured with a pH microsensor. RESULTS: Sufficient contrast-to-noise for producing pHe maps was achieved by using standard image processing methods. A comparison of pHe values measured with acidoCEST MRI and a pH microsensor showed that acidoCEST MRI measured tumor pHe with an accuracy of 0.034 pH units. The MCF-7 tumor model was found to be more acidic compared to the MDA-MB-231 tumor model. The pHe was not related to tumor size during the longitudinal study. CONCLUSIONS: These results show that acidoCEST MRI can create pixelwise tumor pHe maps of mouse models of cancer.

Assessment of carbonic anhydrase IX expression and extracellular pH in B-cell lymphoma cell line models.

The expression of carbonic anhydrase IX (CA IX) and its relationship to acidosis in lymphomas has not been widely studied. We investigated the protein expression of CA IX in a human B-cell lymphoma tissue microarray, and in Raji, Ramos and Granta 519 lymphoma cell lines and tumor models, while also investigating the relationship with hypoxia. An imaging method, acidoCEST magnetic resonance imaging (MRI), was used to estimate lymphoma xenograft extracellular pH (pHe). Our results showed that clinical lymphoma tissues and cell line models in vitro and in vivo had moderate CA IX expression. Although in vitro studies showed that CA IX expression was induced by hypoxia, in vivo studies did not show this correlation. Untreated lymphoma xenograft tumor pHe had acidic fractions, and an acidity score was qualitatively correlated with CA IX expression. Therefore, CA IX is expressed in B-cell lymphomas and is qualitatively correlated with extracellular acidosis in xenograft tumor models.

The reciprocal linear QUEST analysis method facilitates the measurements of chemical exchange rates with CEST MRI.

Magnetic resonance imaging (MRI) contrast media that are detected via chemical exchange saturation transfer (CEST) often require an accurate estimation of their chemical exchange rate, kex . A variety of analysis methods have been proposed to estimate kex , including the nonlinear QUEST analysis method that evaluates the CEST amplitude as a function of saturation time. We have derived a linear version of QUEST, termed the Reciprocal Linear QUEST (RL-QUEST) method. Our simulations and experimental results show that RL-QUEST performs as well as QUEST, while providing a more simplistic fitting procedure. Although CEST results should be acquired with saturation power that has a nutation rate that is faster than kex of the CEST agent, an exact determination of the saturation power is not required to accurately estimate kex with RL-QUEST. This new analysis method requires a determination of the CEST agent's concentration, which is straightforward for the analysis of CEST agents in chemical solutions, but may be a limitation during in vivo CEST MRI studies. Based on the results of this study and previous studies, we provide recommendations for the linear analysis method that should be employed for each type of CEST MRI study.

Evaluations of extracellular pH within in vivo tumors using acidoCEST MRI.

PURPOSE: A practical, noninvasive method is needed to measure the extracellular pH (pHe) within in vivo tumors to longitudinally monitor tumor acidosis. We have optimized a biomedical imaging method, termed acidoCEST MRI, to provide noninvasive assessments of tumor pHe in preclinical models of mammary carcinoma. METHODS: A CEST-FISP MRI method was optimized to detect the chemical exchange saturation transfer (CEST) of two amide protons of a clinically approved CT contrast agent, iopromide. The ratio of the two CEST effects was used to measure pH. Routes of administration of iopromide were evaluated to ensure sufficient delivery of the agent to the tumor. The optimized acidoCEST MRI method was then used to evaluate the change in tumor pHe following alkalinizing bicarbonate treatment. RESULTS: The acidoCEST MRI protocol measured pH between 6.2 and 7.2 pH units. Greater delivery of iopromide was shown to improve the precision of the measurement of tumor pHe, but the agent did not influence the tumor pHe. AcidoCEST MRI was used to longitudinally monitor the effect of bicarbonate treatment on the pHe of tumors and bladders. CONCLUSION: This study demonstrates that an optimized acidoCEST MRI method is a practical, noninvasive method for assessing changes in tumor acidosis.

The Hanes-Woolf linear QUESP method improves the measurements of fast chemical exchange rates with CEST MRI.

PURPOSE: Contrast agents for chemical exchange saturation transfer MRI often require an accurate measurement of the chemical exchange rate. Many analysis methods have been reported that measure chemical exchange rates. Additional analysis methods were derived as part of this study. This report investigated the accuracy and precision of each analysis method. METHODS: Chemical exchange saturation transfer spectra were simulated using the Bloch-McConnell equations modified for chemical exchange. Chemical exchange saturation transfer spectra of iopromide were obtained with a range of saturation times, saturation powers, and concentrations. These simulated and experimental results were used to estimate the chemical exchange rate using the QUESP, QUEST, Omega Plot (LB-QUESP), EH-QUESP, HW-QUESP, LB-Conc, EH-Conc, and HW-Conc methods. RESULTS: Bloch fitting produced the most precise estimates of chemical exchange rates, although substantial expertise and computation time were required to achieve these results. Of the more simplistic analysis methods, the HW-QUESP method produced the most accurate and precise estimates of fast exchange rates. The QUEST and LB-QUESP methods produced the most accurate estimates of slow exchange rates, especially with samples that have short T(1w) relaxation times. CONCLUSIONS: HW-QUESP is a simplistic analysis method that should be used when fast chemical exchange rates need to be estimated from chemical exchange saturation transfer MRI results.

Measuring in vivo tumor pHe with CEST-FISP MRI.

Paramagnetic chemical exchange saturation transfer (PARACEST) MRI contrast agents have been developed that can measure pH in solution studies, but these agents have not previously been detected in vivo. To use the PARACEST agent Yb-DO3A-oAA to measure the extracellular pH (pHe) in tumor tissue, a chemical exchange saturation transfer fast imaging with steady state precession MRI protocol was developed, the saturation period was optimized for sensitive chemical exchange saturation transfer (CEST) detection, and median filtering was used to remove artifacts in CEST spectra. These improvements were used to correlate pH with a ratio of two CEST effects of Yb-DO3A-oAA at a 7 T magnetic field strength (R(2) = 0.99, standard deviation of precision = 0.011 pH units). The PARACEST agent could not be detected in tumor tissue following i.v. injection due to the low sensitivity of in vivo CEST MRI. Yb-DO3A-oAA was detected in tumor tissue and leg muscle after directly injecting the PARACEST agent into these tissues. The measured CEST effects were used to measure a tumor pH of 6.82 ± 0.21 and a leg muscle pH of 7.26 ± 0.14, and parametric pH maps were also generated from these tissue regions. These results demonstrated that tumor pHe can be measured with a PARACEST agent and a rapid CEST-MRI protocol.

Conformational States and kinetics of the calcium binding domain of NADPH oxidase 5.

Superoxide generated by human NADPH oxidase 5 (NOX5) is of growing importance for various physiological and pathological processes. The activity of NOX5 appears to be regulated by a self-contained Ca(2+) binding domain (CaBD). Recently Bánfi et al. suggest that the conformational change of CaBD upon Ca(2+) binding is essential for domain-domain interaction and superoxide production. The authors studied its structural change using intrinsic Trp fluorescence and hydrophobic dye binding; however, their conformational study was not thorough and the kinetics of metal binding was not demonstrated. Here we generated the recombinant CaBD and an E99Q/E143Q mutant to characterize them using fluorescence spectroscopy. Ca(2+) binding to CaBD induces a conformational change that exposes hydrophobic patches and increases the quenching accessibilities of its Trp residues and AEDANS at Cys107. The circular dichroism spectra indicated no significant changes in the secondary structures of CaBD upon metal binding. Stopped-flow spectrometry revealed a fast Ca(2+) dissociation from the N-terminal half, followed by a slow Ca(2+) dissociation from the C-terminal half. Combined with a chemical stability study, we concluded that the C-terminal half of CaBD has a higher Ca(2+) binding affinity, a higher chemical stability, and a slow Ca(2+) dissociation. The Mg(2+)-bound CaBD was also investigated and the results indicate that its structure is similar to the apo form. The rate of Mg(2+) dissociation was close to that of Ca(2+) dissociation. Our data suggest that the N- and C-terminal halves of CaBD are not completely structurally independent.