Claire H. Mitchell

CATSPER Channel-Mediated Ca2+ Entry into Mouse Sperm Triggers a Tail-to-Head Propagation

Abstract Many Ca2+ channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca2+ entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca2+ entry also triggers a tail-to-head Ca2+ propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca2+ concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca2+ propagation through the midpiece leads to a Ca2+-dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca2+ influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca2+ propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.

Release of ATP by a human retinal pigment epithelial cell line: potential for autocrine stimulation through subretinal space

1 Stimulation of purinergic receptors on retinal pigment epithelial (RPE) cells can increase the rate of fluid transport or decrease phagocytosis. This study aims to: determine whether the purine ATP can be released from RPE cells, begin probing the mechanism of any release and test whether cells degrade ATP extracellularly. 2 ATP release was monitored from cultured human ARPE‐19 cells with the luciferin‐luciferase assay. Biphasic release of ATP was triggered by basic fibroblast growth factor (bFGF), by the pyrimidine uridine triphosphate (UTP) and by hypotonicity. 3 The Cl− channel blocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB) inhibited release of ATP, suggesting that release was associated with Cl− channels. 4 Elevating intracellular Ca2+ directly with ionomycin was insufficient to trigger ATP release. 5 UTP induced a biphasic elevation in intracellular Ca2+. NPPB inhibited the second phase, suggesting autostimulation by released ATP. 6 Cells grown on permeable supports showed apical release of ATP, analogous to release into subretinal space in vivo. 7 The presence of ecto‐ATPases on ARPE‐19 cell membranes was suggested by the degradation of ATP added to intact cells. 8 Phagocytosis of fluorescent beads was inhibited by ATP, but the ecto‐5′‐nucleotidase inhibitor α, β‐methylene ADP prevented this, suggesting that inhibition was mediated by extracellular conversion of ATP to adenosine. 9 These results suggest that growth factors, pyrimidines and changes in tonicity could trigger ATP release into subretinal space. The levels of ATP released may be capable of autocrine stimulation of ATP receptors, while conversion to adenosine by ecto‐enzymes could alter phagocytosis.

Elevated Pressure Triggers a Physiological Release of ATP from the Retina: Possible Role for Pannexin Hemichannels

Increased hydrostatic pressure can damage neurons, although the mechanisms linking pressure to neurochemical imbalance or cell injury are not fully established. Throughout the body, mechanical perturbations such as shear stress, cell stretching, or changes in pressure can lead to excessive release of ATP. It is thus possible that increased pressure across neural tissues triggers an elevated release of ATP into extracellular space. As stimulation of the P2X(7) receptor for ATP on retinal ganglion cells leads to elevation of intracellular calcium and excitotoxic death, we asked whether increased levels of extracellular ATP accompanied an elevation in pressure across the retina. The hydrostatic pressure surrounding bovine retinal eyecups was increased and the ATP content of the vitreal compartment adjacent to the retina was determined. A step increase of only 20 mm Hg induced a threefold increase in the vitreal ATP concentration. The ATP levels correlated closely with the degree of pressure increase over 20-100 mm Hg. The increase was transient at lower pressures but sustained at higher pressures. The rise in vitreal ATP was the same regardless of whether nitrogen or air was used to increase pressure, implying changes in oxygen partial pressure did not contribute. Lactate dehydrogenase activity was not affected by pressure, ruling out a substantial contribution from cell lysis. The ATP increase was largely inhibited by either 30 muM 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or 10 muM carbenoxolone (CBX). While this pharmacological profile is consistent with physiological release of ATP through pannexins hemichannels, a contribution from anion channels, vesicular release or other mechanisms cannot be ruled out. In conclusion, a step elevation in pressure leads to a physiologic increase in the levels of extracellular ATP bathing retinal neurons. This excess extracellular ATP may link increased pressure to the death of ganglion cells in acute glaucoma, and suggests a possible role for ATP in the neuronal damage accompanying increased intracranial pressure.

Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors.

•  Neurons can be damaged when tissues are stretched or swollen; while astrocytes can contribute to this process, the mechanosensitive response from neurons is unclear. •  We show here that isolated retinal ganglion cell neurons respond to mechanical strain with a rapid, sustained release of the neurotransmitter ATP. •  The conduit for ATP release was through pannexin hemichannels, with probenicid, carbenoxelone and 10panx inhibiting release. •  Once released, this ATP acts back on the neurons to autostimulate lethal P2X7 receptors, as A438079, AZ 10606120 and zinc reduced currents in whole cell patch clamp recordings. •  Blocking release of ATP through pannexin channels, or activation of P2X7 receptors, might be neuroprotective for stretched or swollen neurons. •  Stretch‐dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X7 receptors, provides a new pathway by which neuronal activity and health can be altered by mechanical strain independently of glial activity.

Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4-/-Mice : Pharmacologic Approaches and Functional Recovery

PURPOSE Degradation of engulfed material is primarily mediated by lysosomal enzymes that function optimally within a narrow range of acidic pH values. RPE cells are responsible for daily degradation of photoreceptor outer segments and are thus particularly susceptible to perturbations in lysosomal pH. The authors hypothesized that elevated lysosomal pH levels could slow enzyme activity and encourage accumulation of partially digested material. Consequently, treatment to lower perturbed lysosomal pH levels may enhance degradative activity. METHODS A high-throughput screening assay was developed to quantify the lysosomal pH of fresh mouse and cultured ARPE-19 cells. The effect of lysosomal pH on outer segment clearance was determined. RESULTS Lysosomal pH is elevated in RPE cells from ABCA4 knockout mice and in cultured human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine. The lysosomal pH of fresh RPE cells from ABCA4(-/-) mice and of chemically compromised RPE cells was reacidified by elevating intracellular cAMP directly. Compromised lysosomal pH was also restored by stimulating A(2A) adenosine or beta-adrenergic receptors, consistent with G(s)-protein coupling of these receptors. Restoring lysosomal pH with these treatments enhanced photoreceptor outer segment clearance, demonstrating functional relevance consistent with an enhancement of degradative enzyme activity. CONCLUSIONS Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function.

Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

Presenilin 1 (PS1) deletion or Alzheimers disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

Mechanosensitive release of adenosine 5′-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: A mechanism for purinergic involvement in chronic strain

As adenosine 5′‐triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1–3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1−/− mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling‐induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg‐MYOCY437H mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma. GLIA 2014;62:1486–1501

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2‐chloro‐N6‐(3‐iodobenzyl)‐adenosine‐5′‐N‐methyluronimide (Cl‐IB‐MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3‐Ethyl‐5‐benzyl‐2‐methyl‐4‐phenylethynyl‐6‐phenyl‐1,4‐(±)‐dihydropyridine‐3,5‐dicarboxylate (MRS‐1191) reduced the protective effects of adenosine. Both Cl‐IB‐MECA and a second A3 receptor agonist IB‐MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPγS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.

From Mechanosensitivity to Inflammatory Responses: New Players in the Pathology of Glaucoma

Abstract Purpose of the study: Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca2+ influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses. Conclusions: Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca2+-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.

Purines in the eye: recent evidence for the physiological and pathological role of purines in the RPE, retinal neurons, astrocytes, Müller cells, lens, trabecular meshwork, cornea and lacrimal gland.

This review highlights recent findings that describ how purines modulate the physiological and pathophysiological responses of ocular tissues. For example, in lacrimal glands the cross-talk between P2X7 receptors and both M3 muscarinic receptors and α1D-adrenergic receptors can influence tear secretion. In the cornea, purines lead to post-translational modification of EGFR and structural proteins that participate in wound repair in the epithelium and influence the expression of matrix proteins in the stroma. Purines act at receptors on both the trabecular meshwork and ciliary epithelium to modulate intraocular pressure (IOP); ATP-release pathways of inflow and outflow cells differ, possibly permitting differential modulation of adenosine delivery. Modulators of trabecular meshwork cell ATP release include cell volume, stretch, extracellular Ca(2+) concentration, oxidation state, actin remodeling and possibly endogenous cardiotonic steroids. In the lens, osmotic stress leads to ATP release following TRPV4 activation upstream of hemichannel opening. In the anterior eye, diadenosine polyphosphates such as Ap4A act at P2 receptors to modulate the rate and composition of tear secretion, impact corneal wound healing and lower IOP. The Gq11-coupled P2Y1-receptor contributes to volume control in Müller cells and thus the retina. P2X receptors are expressed in neurons in the inner and outer retina and contribute to visual processing as well as the demise of retinal ganglion cells. In RPE cells, the balance between extracellular ATP and adenosine may modulate lysosomal pH and the rate of lipofuscin formation. In optic nerve head astrocytes, mechanosensitive ATP release via pannexin hemichannels, coupled with stretch-dependent upregulation of pannexins, provides a mechanism for ATP signaling in chronic glaucoma. With so many receptors linked to divergent functions throughout the eye, ensuring the transmitters remain local and stimulation is restricted to the intended target may be a key issue in understanding how physiological signaling becomes pathological in ocular disease.