As a representative two-dimensional (2D) nanomaterial, graphene oxide (GO) has shown high potential in many applications due to its large surface area, high flexibility, and excellent dispersibility in aqueous solutions. These properties make GO an ideal candidate for bio-imaging, drug delivery, and cancer therapy. When delivered to the body, GO has been shown to accumulate in the liver, the primary accumulation site of systemic delivery or secondary spread from other uptake sites, and induce liver toxicity. However, the contribution of the GO physicochemical properties and individual liver cell types to this toxicity is unclear due to property variations and diverse cell types in the liver. Herein, we compare the effects of GOs with small (GO-S) and large (GO-L) lateral sizes in three major cell types in liver, Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and hepatocytes. While GOs induced cytotoxicity in KCs, they induced significantly less toxicity in LSECs and hepatocytes. For KCs, we found that GOs were phagocytosed that triggered NADPH oxidase mediated plasma membrane lipid peroxidation, which leads to PLC activation, calcium flux, mitochondrial ROS generation, and NLRP3 inflammasome activation. The subsequent caspase-1 activation induced IL-1β production and GSDMD-mediated pyroptosis. These effects were lateral size-dependent with GO-L showing stronger effects than GO-S. Amongst the liver cell types, decreased cell association and the absence of lipid peroxidation resulted in low cytotoxicity in LSECs and hepatocytes. Using additional GO samples with different lateral sizes, surface functionalities, or thickness, we further confirmed the differential cytotoxic effects in liver cells and the major role of GO lateral size in KUP5 pyroptosis by correlation studies. These findings delineated the GO effects on cellular uptake and cell death pathways in liver cells, and provide valuable information to further evaluate GO effects on the liver for biomedical applications.

Epilepsy is characterized by recurrent seizures due to abnormal hyperexcitation of neurons. Recent studies have suggested that the imbalance of excitation and inhibition (E/I) in the central nervous system is closely implicated in the etiology of epilepsy. In the brain, GABA is a major inhibitory neurotransmitter and plays a pivotal role in maintaining E/I balance. As such, altered GABAergic inhibition can lead to severe E/I imbalance, consequently resulting in excessive and hypersynchronous neuronal activity as in epilepsy. Phospholipase C (PLC) is a key enzyme in the intracellular signaling pathway and regulates various neuronal functions including neuronal development, synaptic transmission, and plasticity in the brain. Accumulating evidence suggests that neuronal PLC is critically involved in multiple aspects of GABAergic functions. Therefore, a better understanding of mechanisms by which neuronal PLC regulates GABAergic inhibition is necessary for revealing an unrecognized linkage between PLC and epilepsy and developing more effective treatments for epilepsy. Here we review the function of PLC in GABAergic inhibition in the brain and discuss a pathophysiological relationship between PLC and epilepsy.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P1, S1P2, and S1P3 receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to Gq. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα13 but not Gα12, Gαq, or Gαi. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P3 in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P3 knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P3 KO mouse heart. CYM-51736, an S1P3-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P3 receptor- and Gα13-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P3 receptors could provide therapeutic benefits in ischemic heart disease.

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.

Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. This article is part of a Special Issue entitled Phosphoinositides.

This review discusses the role that the sperm-specific phospholipase C zeta (PLCζ) is proposed to play during the fertilization of mammalian eggs. At fertilization, the sperm initiates development by causing a series of oscillations in cytosolic concentrations of calcium [Ca(2)] within the egg. PLCζ mimics the sperm at fertilization, causing the same pattern of Ca(2+) release as seen at fertilization. Introducing PLCζ into mouse eggs also mimics a number of other features of the way in which the fertilizing sperm triggers Ca(2+) oscillations. We discuss the localization of PLCζ within the egg and present a hypothesis about the localization of PLCζ within the sperm before the initiation of fertilization.

Significant evidence now supports the assertion that cytosolic calcium oscillations during fertilization in mammalian eggs are mediated by a testis-specific phospholipase C (PLC), termed PLC-zeta (PLCζ) that is released into the egg following gamete fusion. Herein, we describe the current paradigm of PLCζ in this fundamental biological process, summarizing recent important advances in our knowledge of the biochemical and physiological properties of this enzyme. We describe the data suggesting that PLCζ has distinct features amongst PLCs enabling the hydrolysis of its substrate, phosphatidylinositol 4,5-bisphosphate (PIP2) at low Ca(2+) levels. PLCζ appears to be unique in its ability to target PIP2 that is present on intracellular vesicles. We also discuss evidence that PLCζ may be a significant factor in human fertility with potential therapeutic capacity.