Shigeru Morishima

Receptor‐mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re‐adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate‐conductance Ca2+‐dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C‐mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein‐coupled Ca2+‐sensing receptor (CaR) enhances the activity of volume‐sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling‐induced and P2Y2 receptor‐mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume‐regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108‐15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be abolished by prior treatment with a blocker of volume‐regulatory K+ or Cl− channels, suggesting that AVD is caused by normotonic activation of ion channels that are normally involved in RVD under hypotonic conditions. Therefore, it is likely that G protein‐coupled receptors involved in RVD regulation and death receptors triggering AVD may share common downstream signals which should give us key clues to the detailed mechanisms of volume regulation and survival of animal cells. In this Topical Review, we look at the physiological ionic mechanisms of cell volume regulation and cell death‐associated volume changes from the facet of receptor‐mediated cellular processes.

Phloretin differentially inhibits volume-sensitive and cyclic AMP-activated, but not Ca-activated, Cl(-) channels.

Some phenol derivatives are known to block volume‐sensitive Cl− channels. However, effects on the channel of the bisphenol phloretin, which is a known blocker of glucose uniport and anion antiport, have not been examined. In the present study, we investigated the effects of phloretin on volume‐sensitive Cl− channels in comparison with cyclic AMP‐activated CFTR Cl− channels and Ca2+‐activated Cl− channels using the whole‐cell patch‐clamp technique. Extracellular application of phloretin (over 10 μM) voltage‐independently, and in a concentration‐dependent manner (IC50 ∼30 μM), inhibited the Cl− current activated by a hypotonic challenge in human epithelial T84, Intestine 407 cells and mouse mammary C127/CFTR cells. In contrast, at 30 μM phloretin failed to inhibit cyclic AMP‐activated Cl− currents in T84 and C127/CFTR cells. Higher concentrations (over 100 μM) of phloretin, however, partially inhibited the CFTR Cl− currents in a voltage‐dependent manner. At 30 and 300 μM, phloretin showed no inhibitory effect on Ca2+‐dependent Cl− currents induced by ionomycin in T84 cells. It is concluded that phloretin preferentially blocks volume‐sensitive Cl− channels at low concentrations (below 100 μM) and also inhibits cyclic AMP‐activated Cl− channels at higher concentrations, whereas phloretin does not inhibit Ca2+‐activated Cl− channels in epithelial cells.

Volume-sensitive chloride channels in mouse cortical neurons: characterization and role in volume regulation

Because persistent swelling causes cell damage and often results in cell death, volume regulation is an important physiological function in both neuronal and non‐neuronal cells. Brain cell swelling has been observed not only in various pathological conditions but also during physiological synaptic transmissions. Volume‐sensitive anion channels have been reported to play an important role in the regulatory volume decrease occurring after osmotic swelling in many cell types. In this study, using a two‐photon laser scanning microscope and patch‐clamp techniques, we found that mouse cortical neurons in primary culture exhibit regulatory volume decrease after transient swelling and activation of Cl– currents during exposure to a hypotonic solution. The regulatory volume decrease was inhibited by Cl– channel blockers or K+ channel blockers. Swelling‐activated Cl– currents exhibited outward rectification, time‐dependent inactivation at large positive potentials, a low‐field anion permeability sequence, an intermediate unitary conductance and sensitivity to known blockers of volume‐sensitive Cl– channels. Thus, it is concluded that the activity of the volume‐sensitive outwardly rectifying Cl– channel plays a role in the control of cell volume in cortical neurons.

Snapin, a New Regulator of Receptor Signaling, Augments α1A-Adrenoceptor-operated Calcium Influx through TRPC6

Activation of Gq-protein-coupled receptors, including the α1A-adrenoceptor (α1A-AR), causes a sustained Ca2+ influx via receptor-operated Ca2+ (ROC) channels, following the transient release of intracellular Ca2+. Transient receptor potential canonical (TRPC) channel is one of the candidate proteins constituting the ROC channels, but the precise mechanism linking receptor activation to increased influx of Ca2+ via TRPCs is not yet fully understood. We identified Snapin as a protein interacting with the C terminus of the α1A-AR. In receptor-expressing PC12 cells, co-transfection of Snapin augmented α1A-AR-stimulated sustained increases in intracellular Ca2+ ([Ca2+]i) via ROC channels. By altering the Snapin binding C-terminal domain of the α1A-AR or by reducing cellular Snapin with short interfering RNA, the sustained increase in [Ca2+]i in Snapin-α1A-AR co-expressing PC12 cells was attenuated. Snapin co-immunoprecipitated with TRPC6 and α1A-AR, and these interactions were augmented upon α1A-AR activation, increasing the recruitment of TRPC6 to the cell surface. Our data suggest a new receptor-operated signaling mechanism where Snapin links the α1A-AR to TRPC6, augmenting Ca2+ influx via ROC channels.

Ca2+-sensing receptor-mediated regulation of volume-sensitive Cl− channels in human epithelial cells

1 Since extracellular Ca2+ or Mg2+ has been reported to modulate swelling‐activated Cl− currents, we examined the expression of the G protein‐coupled Ca2+‐sensing receptor (CaR) and its involvement in the regulation of volume‐sensitive Cl− channels in a human epithelial cell line (Intestine 407). 2 Reverse transcriptase‐polymerase chain reaction and immunoblotting analysis showed that Intestine 407 cells express CaR mRNA and protein. 3 The swelling‐activated whole‐cell Cl− current was voltage‐independently augmented by extracellular Ca2+ or Mg2+. In addition, Ca2+ or Mg2+ voltage‐dependently accelerated the inactivation kinetics of the Cl− current. 4 Neomycin, spermine and La3+ augmented volume‐sensitive Cl− currents. However, these CaR agonists failed to affect depolarization‐induced inactivation. 5 Intracellular application of GTPγS, but not GDPβS, increased the amplitude of the swelling‐induced Cl− current without affecting the basal current. The upregulating effect of Ca2+ on the Cl− current amplitude was abolished by either GTPγS or GDPβS. In contrast, GTPγS and GDPβS failed to affect the inactivation kinetics of the Cl− current and the accelerating effect of Ca2+ thereon. 6 The Cl− current amplitude was enlarged by stimulation with forskolin, dibutyryl cAMP and IBMX. During the cAMP stimulation, extracellular Ca2+ failed to increase the Cl− current but did accelerate depolarization‐induced inactivation. 7 It is concluded that stimulation of the CaR induces upregulation of volume‐sensitive Cl− channels via a G protein‐mediated increase in intracellular cAMP in the human epithelial cell. However, the accelerating effect of extracellular divalent cations on the inactivation kinetics of the Cl− current is induced by a mechanism independent of the CaR and cAMP.

Identification of the α1L-adrenoceptor in rat cerebral cortex and possible relationship between α1L- and α1A-adrenoceptors

In addition to α1A, α1B and α1D‐adrenoceptors (ARs), putative α1L‐ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the α1A‐AR and clarify its pharmacological profile using a radioligand binding assay.

Recovery from lactacidosis‐induced glial cell swelling with the aid of exogenous anion channels

Hypotonic challenge induces transient swelling in glial cells, which is typically followed by a regulatory volume decrease (RVD). In contrast, lactic acidosis (lactacidosis) induces persistent cell swelling in astrocytes without an accompanying RVD. In the present study, we studied the mechanisms by which lactacidosis interferes with normal volume regulation in rat astrocytic glioma C6 cells. Following exposure of C6 cells to a hypotonic challenge, a current was detected that exhibited properties consistent with those of volume‐sensitive outwardly rectifying (VSOR) anion channels. When exposed to in vitro conditions designed to simulate lactacidosis, C6 cells failed to respond to hypotonic stress with an RVD, and VSOR anion currents were not activated. When added to C6 cells, an anion channel‐forming protein purified from Helicobacter pylori, VacA, was found to form anion‐selective channels in the plasma membrane, and the activity of the VacA channel was not affected by lactacidosis (pH 6.2). Cells preincubated with VacA and then exposed to lactacidotic conditions underwent transient swelling followed by RVD. In contrast, application of a cation ionophore, gramicidin, failed to inhibit lactacidosis‐induced persistent cell swelling. From these results, we conclude that inhibition of a volume‐sensitive anion channel contributes to persistent swelling induced by lactacidosis in glial cells. Introduction of anion channel activity into glial cells might provide a novel approach for treating cerebral edema, which is associated with lactacidosis in cerebral ischemia or head injury. GLIA 41:247–259, 2003.

Impaired activity of volume-sensitive anion channel during lactacidosis-induced swelling in neuronally differentiated NG108-15 cells

Acidosis coupled to lactate accumulation, called lactacidosis, occurs in cerebral ischemia or trauma and is known to cause persistent swelling in neuronal and glial cells. It is therefore possible that mechanisms of cell volume regulation are impaired during lactacidosis. Here we tested this possibility using neuronally differentiated NG108-15 cells. These cells responded to a hypotonic challenge with osmotic swelling followed by a regulatory volume decrease (RVD) under physiological pH conditions in the absence of lactate. Under normotonic conditions, sustained cell swelling without subsequent RVD was induced by exposure to lactate-containing solution with acidic pH (6.4 or 6.2), but not with physiological pH (7.4). Under whole-cell patch-clamp, osmotic swelling was found to activate outwardly rectifying Cl(-) currents in cells exposed to control hypotonic solution. A Cl(-) channel blocker, NPPB, inhibited both RVD and the swelling-activated Cl(-) current. RVD and the volume-sensitive Cl(-) current were also markedly inhibited by lactacidosis (pH 6.4 or 6.2), but neither by application of lactate with physiological pH (7.4) nor by acidification without lactate (pH 6.2). RT-PCR analysis showed mRNA expression of two isoforms of proton-coupled monocarboxylate transporters, MCT1 and MCT8, in differentiated NG108-15 cells. Thus, we conclude that persistence of neuronal cell swelling under lactacidosis is coupled to an impairment of the activity of the volume-sensitive Cl(-) channel and to dysfunction of RVD. It is also suggested that the volume-sensitive Cl(-) channel is inhibited by intracellular acidification induced by MCT-mediated proton influx under lactacidosis.

Visualization and Tissue Distribution of α1L-Adrenoceptor in Human Prostate by the Fluorescently Labeled Ligand Alexa-488-Silodosin

PURPOSE Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression. MATERIALS AND METHODS Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. RESULTS Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. CONCLUSIONS Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.

Native profiles of α1A‐adrenoceptor phenotypes in rabbit prostate

α1‐Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of α1‐adrenoceptor phenotypes and to clarify their relationship.