Impact of P2X Purinoceptors on Goblet Cell Function: Implications for Dry Eye.

By providing ~70% of the eye's refractive power, the preocular tear film is essential for optimal vision. However, its integrity is often jeopardized by environmental and pathologic conditions that accelerate evaporation and cause sight-impairing dry eye. A key adaptive response to evaporation-induced tear film hyperosmolarity is the reflex-triggered release of tear-stabilizing mucin from conjunctival goblet cells.

Bioelectric Responses of Conjunctival Goblet Cells to Dry Eye: Impact of Ion Channels on Exocytotic Function and Viability.

How ion channels impact the response of the ocular surface to dry eye is only beginning to be explored. Here, we review recent progress and provide new experimental data clarifying the exocytosis-altering actions of ion channels in conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to the pre-ocular hyperosmolarity that characterizes dry eye. Patch-clamp recordings of goblet cells located in freshly excised rat conjunctiva reveal that these mucin-releasing cells respond to sustained hyperosmolarity by sequentially activating their ATP-sensitive potassium (K), nonspecific cation (NSC), voltage-gated calcium (VGCC), and P2X channels; each of which modulates exocytosis.

Treatment with shCCL20-CCR6 nanodendriplexes and human mesenchymal stem cell therapy improves pathology in mice with repeated traumatic brain injury.

Traumatic brain injury (TBI) is a devastating neurological disorder, although the underlying pathophysiology is poorly understood. TBI causes blood-brain barrier (BBB) disruption, immune cell trafficking, neuroinflammation and neurodegeneration. CCL20 is an important chemokine mediating neuroinflammation.

How goblet cells respond to dry eye: adaptive and pathological roles of voltage-gated calcium channels and P2X purinoceptors.

Dry eye is a common sight-impairing, painful disorder characterized by disruption of the preocular tear film, whose integrity is required for ~70% of the eye's refractive power. A universal feature of clinical dry eye is hyperosmolarity of the tears resulting from their accelerated evaporation due to dysfunction of tear- and oil-producing ocular glands. A key adaptive response to dryness/hyperosmolarity is release of tear-stabilizing mucin by conjunctival goblet cells.

Electrotonic transmission in the retinal vasculature: inhibitory role of the diabetes/VEGF/aPKC pathway.

The deleterious impact of diabetes on the retina is a leading cause of vision loss. Ultimately, the hypoxic retinopathy caused by diabetes results in irreversible damage to vascular, neuronal, and glial cells. Less understood is how retinal physiology is altered early in the course of diabetes.

Purinergic Vasotoxicity: Role of the Pore/Oxidant/K Channel/Ca Pathway in P2X-Induced Cell Death in Retinal Capillaries.

P2X receptor/channels in the retinal microvasculature not only regulate vasomotor activity, but can also trigger cells in the capillaries to die. While it is known that this purinergic vasotoxicity is dependent on the transmembrane pores that form during P2X activation, events linking pore formation with cell death remain uncertain. To better understand this pathophysiological process, we used YO-PRO-1 uptake, dichlorofluorescein fluorescence, perforated-patch recordings, fura-2 imaging and trypan blue dye exclusion to assess the effects of the P2X agonist, benzoylbenzoyl-ATP (BzATP), on pore formation, oxidant production, ion channel activation, [Ca] and cell viability.

Role of ion channels in the functional response of conjunctival goblet cells to dry eye.

Optimal vision requires an ocular surface with a stable tear film whose many critical tasks include providing >70% of the eye's refractive power. However, for millions, tear film instability produces uncomfortable sight-impairing dry eye. Despite the multitude of etiologies for dry eye, a universal hallmark is hyperosmolarity of the tear film.

Bioelectric impact of pathological angiogenesis on vascular function.

Pathological angiogenesis, as seen in many inflammatory, immune, malignant, and ischemic disorders, remains an immense health burden despite new molecular therapies. It is likely that further therapeutic progress requires a better understanding of neovascular pathophysiology. Surprisingly, even though transmembrane voltage is well known to regulate vascular function, no previous bioelectric analysis of pathological angiogenesis has been reported.

The electrotonic architecture of the retinal microvasculature: diabetes-induced alteration.

Although microvascular cell death is a well established hallmark of diabetic retinopathy, which is a major cause of vision loss, much remains to be learned about the functional changes that precede the onset of morphological damage to retinal blood vessels. Early alterations of function are of interest since they may contribute to the development of irreversible pathological events. Because one of the earliest retinal effects of diabetes is the dysregulation of blood flow, we asked whether diabetes alters the functional organization of the capillary/arteriolar complex, which is the operational unit that plays an important role in regulating local perfusion.

Vulnerability of the retinal microvasculature to oxidative stress: ion channel-dependent mechanisms.

Although oxidative stress is a hallmark of important vascular disorders such as diabetic retinopathy, it remains unclear why the retinal microvasculature is particularly vulnerable to this pathophysiological condition. We postulated that redox-sensitive ion channels may play a role. Using H(2)O(2) to cause oxidative stress in microvascular complexes freshly isolated from the adult rat retina, we assessed ionic currents, cell viability, intracellular oxidants, and cell calcium by using perforated-patch recordings, trypan blue dye exclusion, and fura-2 fluorescence, respectively.

Retinovascular physiology and pathophysiology: new experimental approach/new insights.

An important challenge in visual neuroscience is to understand the physiology and pathophysiology of the intra-retinal vasculature, whose function is required for ophthalmoception by humans and most other mammals. In the quest to learn more about this highly specialized portion of the circulatory system, a newly developed method for isolating vast microvascular complexes from the rodent retina has opened the way for using techniques such as patch-clamping, fluorescence imaging and time-lapse photography to elucidate the functional organization of a capillary network and its pre-capillary arteriole. For example, the ability to obtain dual perforated-patch recordings from well-defined sites within an isolated microvascular complex permitted the first characterization of the electrotonic architecture of a capillary/arteriole unit.

Vulnerability of the retinal microvasculature to hypoxia: role of polyamine-regulated K(ATP) channels.

Purpose: It is uncertain why retinal capillaries are particularly vulnerable to hypoxia. In this study, it was hypothesized that their specialized physiology, which includes being the predominant microvascular location of functional adenosine triphosphate-sensitive potassium (K(ATP)) channels, boosts their susceptibility to hypoxia-induced cell death.

Methods: Cell viability, ionic currents, intracellular calcium, and pericyte contractility in microvascular complexes freshly isolated from the rat retina were assessed using trypan blue dye exclusion, perforated-patch recordings, fura-2 fluorescence, and time-lapse videos.

The electrotonic architecture of the retinal microvasculature: modulation by angiotensin II.

The capillary/arteriole complex is the key operational unit regulating local perfusion to meet metabolic demand. However, much remains to be learned about how this multi cellular unit is functionally organized. To help address this challenge, we characterized the electrotonic architecture of the retinal microvasculature, which is particularly well adapted for the decentralized control of blood flow.

Diabetes-induced inhibition of voltage-dependent calcium channels in the retinal microvasculature: role of spermine.

Purpose: Although decentralized control of blood flow is particularly important in the retina, knowledge of the functional organization of the retinal microvasculature is limited. Here, the authors characterized the distribution and regulation of L-type voltage-dependent calcium channels (VDCCs) within the most decentralized operational complex of the retinal vasculature--the feeder vessel/capillary unit--which consists of a capillary network plus the vessel linking it with a myocyte-encircled arteriole.

Methods: Perforated-patch recordings, calcium-imaging, and time-lapse photography were used to assess VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter, in microvascular complexes freshly isolated from the rat retina.

Functional K(ATP) channels in the rat retinal microvasculature: topographical distribution, redox regulation, spermine modulation and diabetic alteration.

The essential task of the circulatory system is to match blood flow to local metabolic demand. However, much remains to be learned about this process. To better understand how local perfusion is regulated, we focused on the functional organization of the retinal microvasculature, which is particularly well adapted for the local control of perfusion.

Rapid, noninvasive detection of diabetes-induced retinal metabolic stress.

Objective: To test whether subjects with diabetes mellitus (DM) have enhanced retinal flavoprotein autofluorescence compared with age-matched control subjects using a rapid, noninvasive clinical imaging method.

Methods: Twenty-one subjects with DM and 21 healthy age-matched control volunteers were subjected to retinal imaging using 1-ms flashes of 467-nm light. Flavoprotein autofluorescence for each flash at 535 nm was recorded using an electron-multiplying charged-coupled device camera with a 512x512-pixel chip.

Loss of insulin-mediated vasoprotection: early effect of diabetes on pericyte-containing microvessels of the retina.

Purpose: Microvascular cell death is a prominent pathologic feature of the retinopathy associated with insulin-deficient diabetes. The aim of this study was to test the hypothesis that reduced insulin action may contribute to microvascular damage in the diabetic retina.

Methods: Microvascular complexes were isolated from retinas of healthy rats and those made insulin deficient by streptozotocin.

Physiology and pathobiology of the pericyte-containing retinal microvasculature: new developments.

Evidence is accumulating that pericyte-containing microvessels, which constitute the largest component of the circulatory system, actively regulate capillary perfusion. Because the retinal vasculature is highly specialized for the local control of blood flow, experimental study of its microvessels is proving useful in the quest to elucidate the mechanisms by which local perfusion is regulated. The microcirculation of the retina is also a focus of considerable attention due to its vulnerability to diabetes, which is a leading cause of vision loss.

NAD+-induced vasotoxicity in the pericyte-containing microvasculature of the rat retina: effect of diabetes.

Purpose: It was recently proposed that activation of P2X(7) purinoceptors may play a role in causing cell death in the pericyte-containing microvasculature of the diabetic retina. This hypothesis is supported by the observation that diabetes enhances lethal pore formation in retinal microvessels exposed to synthetic P2X(7) agonists. The goal of this study was to determine whether purinergic vasotoxicity can be triggered by the endogenous molecule nicotinamide adenosine dinucleotide (NAD(+)), which is a substrate for ecto-ribosylation reactions known to activate P2X(7) receptor/channels in other cell types.

Electrotonic transmission within pericyte-containing retinal microvessels.

Objective: Little is known about the electrotonic architecture of the pericyte-containing retinal microvasculature. Here, the authors focus on the cell-to-cell transmission of hyperpolarization, which can induce abluminal pericytes to relax and lumens to dilate.

Methods: With perforated-patch pipettes, the authors monitored the membrane potentials and ionic currents of pairs of pericytes located on freshly isolated rat retinal microvessels.

Topographical heterogeneity of K(IR) currents in pericyte-containing microvessels of the rat retina: effect of diabetes.

Although inwardly rectifying potassium (K(IR)) channels are known to have important functional roles in arteries and arterioles, knowledge of these channels in pericyte-containing microvessels is limited. A working hypothesis is that K(IR) channel activity affects the membrane potential and thereby the contractile tone of abluminal pericytes whose contractions and relaxations may regulate capillary perfusion. Because pericyte function is thought to be particularly important in the retina, we used the perforated-patch technique to monitor the ionic currents of pericytes located on microvessels freshly isolated from the rat retina.

Extracellular lactate as a dynamic vasoactive signal in the rat retinal microvasculature.

We tested the hypothesis that extracellular lactate regulates the function of pericyte-containing retinal microvessels. Although abluminally positioned pericytes appear to adjust capillary perfusion by contracting and relaxing, knowledge of the molecular signals that regulate the contractility of these mural cells is limited. Here, we focused on lactate because this metabolic product is in the retinal extracellular space under both physiological and pathophysiological conditions.

Regulation of P2X7-induced pore formation and cell death in pericyte-containing retinal microvessels.

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X(7) and P2Y(4) purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X(7) receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality.

Effects of angiotensin II on the pericyte-containing microvasculature of the rat retina.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography.

Enhancement of P2X(7)-induced pore formation and apoptosis: an early effect of diabetes on the retinal microvasculature.

Purpose: A sight-threatening complication of diabetes is cell death in retinal capillaries. Currently, the mechanisms responsible for this classic manifestation of diabetic retinopathy remain uncertain. The hypothesis for the current study is that diabetes increases the vulnerability of retinal microvessels to the potentially lethal consequences of having their P2X(7) purinoceptors activated.