Various physiological and pathological processes are accompanied with the local acidification of extracellular local pH. However, imaging tools to investigate the spatio-temporal dynamics as well as the functional significance of cell surface pH are limitedly available. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). PEG-lipid, amphiphilic synthetic polymer, is a biomaterial originally synthesized for cell-surface engineering for transplantation therapy. When added into the cell culture medium, FITC-PEG-lipid was spontaneously inserted into the plasma membrane via its phospholipid moiety. FITC-PEG-lipid was retained at the extracellular surface due to the hydrophobic PEG moiety. The ratiometric readout of FITC fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH (pH 5.0–7.5). The pH measurement with FITC-PEG-lipid was accurate enough to distinguish the difference of 0.1 pH unit for the external solutions at pH 5.9, 6.0 and 6.1, near the inflection point of fluorescence ratio. The response of FITC-PEG-lipid to the extracellular pH was reversible. Continuous alteration of extracellular pH was successfully visualized by time-lapse imaging analysis. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell surface-anchored pH probe. The simple labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.

Gastric proton pump (H⁺,K⁺-ATPase) which is responsible for H⁺ secretion of gastric acid (HCl) in gastric parietal cells is the major therapeutic target for treatment of acid-related diseases. H⁺,K⁺-ATPase consists of two subunits, a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). N-glycosylation of βHK is essential for trafficking and stability of αHK in apical membrane of gastric parietal cells. Terminal sialic acid residues on sugar chains have an important role in various cellular functions. Recently, we succeeded in visualizing the sialylation and desialylation dynamics of βHK using a fluorescence bioimaging nanoprobe consisting of biocompatible polymers conjugated with lectins for detecting sialic acid. In H⁺,K⁺-ATPase-expressing cell lines, rat gastric mucosa, and primary culture of rat gastric parietal cells, fluorescence imaging of sialic acid with the nanoprobe showed that sialylation of βHK is regulated by intragastric pH and that inhibition of gastric acid secretion induces desialylation of βHK. In biochemical and pharmacological studies, we revealed that enzyme activity of αHK is negatively regulated by desialylation of βHK. Our studies uncovered a novel negative-feedback mechanism of H⁺,K⁺-ATPase in which sialic acids of βHK positively regulates H⁺,K⁺-ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of βHK. In this topic, we introduce the overview of our research using the bioimaging nanoprobe.

Scanning electrochemical microscopy (SECM), which utilizes microelectrodes as a probe to measure the chemicals released and consumed by cells as current signal, is a promising tool for measuring the metabolites of cells. We have improved SECM resolution for single cell imaging by miniaturizing the size of the electrode and developing hybrid system of SECM and scanning ion conductance microscopy (SICM), which utilizes nanopipette as a probe to measure live cell topography. SECM-SICM provides simultaneous imaging of concentration profiles of chemical substances and cell surface topography. Using this system, we successfully measured the release of neurotransmitters from PC12 cells. In addition, the nanoscale electrodes are useful for intracellular chemical detection by inserting the electrodes into cells and measured reactive oxygen species (ROS).

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 μm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

The neuronal activity forms the basis of functional circuits and brain functions. To understand how the brain operates, recording of neural activity at micro-, meso-, and macro-scales is required. Recently, improved optical microscopic technology helps us to develop a whole-brain imaging system at a single-cell resolution. The combination of a whole-brain imaging system and a reporter system of neuronal activation enables a whole-brain mapping of neuronal activity. In this review, we first describe the high-speed and scalable whole-brain imaging system including our recently developed system, named FAST, and then present the instances of whole-brain mapping of neuronal activity and its analytical methods.

Primary human hepatocytes are widely used to study drug metabolism and enzyme induction. However, primary hepatocytes rapidly lose their hepatic function in conventional 2D cultures. Recently, a microphysiological system that overcomes this drawback has been actively investigated and applied in drug discovery research. Such novel in vitro models are desirable for the evaluation of the metabolic clearance of drugs with low turnover, drug-induced liver injury, and chronic liver diseases like liver fibrosis. This article reviews the characteristics and recent advances in 3D-bioprinted human liver tissue models in drug discovery research.

Linaclotide (Linzess^® tablets 0.25 mg) is a guanylate cyclase-C (GC-C) agonist with high selectivity and binding affinity to GC-C. In Japan, linaclotide was approved for 〝irritable bowel syndrome with constipation (IBS-C)〟 in December 2016 and 〝chronic constipation (CC) (excluding constipation due to organic disease)〟 in August 2018. Non-clinical studies demonstrated that linaclotide binding to GC-C increases intracellular cyclic guanosine monophosphate (cGMP), resulting in increased fluid secretion and gastrointestinal transit. In rats with colonic hyperalgesia, but not in normal rats, linaclotide suppressed the visceral nociceptive response, mediated by increased submucosal cGMP. In clinical studies in Japan, improvements were observed in the responder rates for global assessment of IBS symptom relief, complete spontaneous bowel movements in patients with IBS-C, and the frequency of spontaneous bowel movement in patients with CC, which were maintained during long-term treatment. Additionally, abdominal bloating, which has been associated with lower quality of life (QOL) and lower satisfaction with other approved therapies, and IBS QOL were improved throughout treatment with linaclotide. Diarrhea, a consequence of linaclotide’s mechanism of action, was observed during the clinical studies, but was generally controllable by decreasing the linaclotide dose. No drug resistance was observed during the clinical studies, unlike some other approved agents. These results of non-clinical and clinical studies demonstrate that linaclotide can improve constipation, various abdominal symptoms, and QOL with a favorable safety profile in patients with IBS-C and CC.