Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia.

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.

Activation of normal and cystic fibrosis Cl- channels by voltage, temperature, and trypsin.

In cystic fibrosis (CF) phosphorylation-dependent activation of outwardly rectifying apical membrane Cl- channels is defective. To further understand regulation of this channel we examined several other mechanisms of channel activation in normal and CF cells. Previous studies have shown that strong membrane depolarization can activate channels in excised cell-free membrane patches. Here we show that such activation is dependent on both the absolute membrane voltage and the duration of depolarization. Moreover, activation was reversible by membrane hyperpolarization. In some cases, excising patches of membrane from the cell caused channel activation, even in the absence of depolarization. However, the frequency of channel activation with patch excision increased when bath temperature was increased from 23 to 37 degrees C. Although the channel remained in the activated state when temperature was reduced to 23 degrees C, subsequent hyperpolarization inactivated the channel. In cell-attached patches, neither depolarization nor increasing bath temperature to 37 degrees C activated channels, suggesting that neither is physiologically important in regulation of the channel. Thus changes in membrane voltage and bath temperature appear to cause a nonenzymatic change in the channel's conformation; the interactions between voltage and temperature suggest that they may affect the same process. To determine if a proteolytic alteration of the channel could also cause activation, we added trypsin to the cytosolic surface of excised membrane patches. Trypsin activated channels, which could not then be inactivated by either hyperpolarization or phosphorylation with PKC, suggesting that trypsin removed or altered a region of the channel involved in inactivation. All of these interventions activated Cl- channels from both normal and CF cells. Thus many aspects of Cl- channel activation are normal in CF; only phosphorylation-dependent activation is defective.

Mikulicz's disease: a new perspective and literature review.

PURPOSE: To report the clinical and pathophysiologic features of two patients with Mikulicz's disease and to further characterize recommendations for diagnosis and management with a review of the literature. METHODS: Retrospective nonrandomized consecutive case series, Jules Stein Eye Institute, David Geffen School of Medicine at UCLA. RESULTS: Mikulicz's disease is characterized by symmetric lacrimal, parotid, and submandibular gland enlargement with associated lymphocytic infiltrations. The authors noted two cases of Mikulicz's disease. The diagnosis of Mikulicz's disease was based on the following criteria: 1) symmetric and persistent swelling of the lacrimal glands and either or both of the major salivary glands (parotid and submandibular); and 2) the exclusion of other diseases that may mimic this presentation, such as sarcoidosis, viral infection, or lymphoproliferative disorders. CONCLUSIONS: Mikulicz's disease is a condition in which there is bilateral lacrimal and salivary gland swelling that is not associated with other systemic conditions. The condition is self-limiting and most often, the diagnosis is a clinical one. Previously, Mikulicz's disease was often considered as a subtype of Sjögren's syndrome (SS). Clinical and immunologic differences between Mikulicz's disease and SS may warrant further consideration of Mikulicz's disease as a specific autoimmune phenomenon separate from SS, and Mikulicz's disease may be amenable to different treatment modalities than those employed in patients with SS.

Neuroleptics antagonize a calcium-activated potassium channel in airway smooth muscle.

We examined the effect of neuroleptics on Ca-activated K channels from dog airway smooth muscle cells. Because these agents inhibit a variety of other Ca-mediated processes, it seemed possible that they might also inhibit Ca-activated K channels. In excised, inside-out patches, several neuroleptics potently and reversibly inhibited the K channel from the internal but not the external surface of the patch. Measurements of the effect on open probability and open- and closed-state durations support a simple kinetic model in which neuroleptics bind to and block the open channel. Inhibition by neuroleptics was moderately voltage dependent, with blockers less potent at hyperpolarizing voltages. The relationship between voltage and the dissociation constant for the blocker suggests that the binding site is one-third of the way across the channel's electrical field. Equilibrium dissociation constants for the drug-channel complex were: haloperidol, 1.0 +/- 0.1 microM; trifluoperazine, 1.4 +/- 0.1 microM; thioridazine, 2.4 +/- 0.1 microM; and chlorpromazine, 2.0 microM. This rank-order potency is different from their potency as calmodulin inhibitors, which suggests that neuroleptics bind to the channel rather than a calmodulin-channel complex.

Identification and regulation of whole-cell chloride currents in airway epithelium.

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.

Lagophthalmos in enophthalmic eyes.

AIMS: To report a case series of enophthalmic patients with lagophthalmos. METHODS: A retrospective review of the electronic medical records at a tertiary health care centre of all patients with the diagnoses of "enophthalmos" and "lagophthalmos". Patients who had a history of diseases (such as Graves' orbitopathy), trauma or surgery of the orbit and eyelid were excluded. Enophthalmos was defined as exophthalmometric reading of 14 mm or less in both eyes. RESULTS: Seven patients (14 eyes) with bilateral enophthalmos were found to have concomitant lagophthalmos. All patients had deep superior sulci bilaterally. The upper eyelids were seen to be severely retro-placed behind the superior orbital rim. The extraocular motilities were full with no focal neurological deficit. The orbicularis oculi function was normal with no facial paralysis. The orbits were soft on retropulsion and no facial asymmetry was noted. The mean exophthalmolmetry reading measured 12.6 (SD 1.1) mm. The lagophthalmos varied from 1-5 mm. One patient (one eye) with 3 mm lagophthalmos developed a corneal ulcer and was treated with topical antibiotics and gold weight placement in the upper eyelid. CONCLUSION: Enophthalmic patients with deep superior sulci and retro-placed upper eyelids may present with lagophthalmos and exposure keratopathy.

Management of severe cicatricial entropion using shared mucosal grafts.

OBJECTIVE: To retrospectively analyze our experience using nasal turbinate and hard palate mucosal grafts as shared buttress grafts between the upper and lower eyelid for reconstruction in severe cicatricial entropion. SURGICAL TECHNIQUES: A horizontal tarsectomy is performed in the upper and lower eyelid approximately 2 mm posterior to the gray line. The distal tarsal segments are then dissected and rotated 180 degrees. A graft of nasal turbinate mucosa or hard palate mucosa measuring 1.5 x 3 cm is harvested. The graft is sutured to the cut edge of tarsus in the upper and lower eyelid. The rotated distal tarsal segment is stabilized against the graft using 5 mattress sutures. After 3 weeks, the graft is split by sharp dissection between the upper and lower eyelids. METHODS: The medical records of 12 consecutive patients, representing 15 shared buttress grafts, were reviewed. There were 5 hard palate and 10 nasal turbinate mucosal grafts placed. Follow-up ranged from 2 months to 7 years. RESULTS: The amount of corneal stipple, as well as subjective patient comfort, improved after eyelid margin reconstruction in 12 of the 15 eyes. One patient's visual acuity improved by more than 2 lines after surgery. There were no cases of failure of graft survival and no complications directly related to the shared graft technique. Recurrent entropion and trichiasis were noted in 3 eyelids more than a year after graft placement, reflecting ongoing cicatrization in these eyelids. Hard palate mucosal grafts were irritating to the corneal surface, requiring removal of the epithelium using a diamond burr and bandage contact lens wear. Nasal turbinate mucosal grafts were better tolerated by the corneal surface and had the added benefit of mucous production. CONCLUSIONS: Eyelid reconstruction using nasal turbinate and hard palate mucosal tissues as a shared buttress graft is a viable treatment option for patients with severe cicatricial entropion. Resolution of trichiasis and mechanical corneal abrasion was noted in 13 (86%) of 15 patients with no specific complications related to the technique. The shared buttress technique successfully autostents the healing eyelid margins, makes good use of the large turbinate mucosal graft, and minimizes trips to the operating room. When the mechanical requirements of eyelid margin reconstruction do not require the sturdiness of hard palate mucosa, nasal turbinate mucosa is a preferable graft tissue because it is better tolerated by the corneal surface and produces mucous.

Phosphorylation-dependent regulation of apical membrane chloride channels in normal and cystic fibrosis airway epithelium.

The observations described herein allow us to make several inferences about PKC and regulation of normal and CF Cl- channels. FIGURE 5 shows a model that summarizes these observations. In this model, for the sake of clarity, we refer to the channel as a single entity, but note that it may consist of multiple subunits and associated proteins. FIGURE 5A shows the channel in an inactivated state following excision from the cell. The channel can be activated by strong membrane depolarization, via an unknown mechanism, or by phosphorylation with PKA or PKC at a low [Ca2+] We speculate that PKA and PKC may phosphorylate and activate the channel at the same site, or region of the channel, because phosphorylation-dependent activation by both is defective in CF. This result suggests that the CF defect might lie in a defective phosphorylation site on the channel, or associated protein, or in the mechanism that converts phosphorylation into a change in channel conformation, such as activation. Activated channels can be inactivated by PKC at a high [Ca2+]. At high [Ca2+], PKC maintains the channel in an inactivated state and it inactivates channels that have been activated by PKC at low [Ca2+], by depolarization, or by PKA. Both activation and inactivation appear to result from phosphorylation; neither can be explained by down-regulation of the channel. There are several possible ways to explain the two opposite effects of PKC on the Cl- channel: different responses may be due to an effect of Ca2+ on the channel, on PKC, or on the interaction between the two.(ABSTRACT TRUNCATED AT 250 WORDS)

Inferior oblique muscle location after enucleation and evisceration.

PURPOSE: To report the location of the inferior oblique muscle after enucleation without primary attachment of the muscle to the orbital implant and after evisceration. METHODS: Interventional case series. Retrospectively, eight orbital magnetic resonance imaging (MRI) studies were analyzed, four after enucleation and four after evisceration, to assess the position of the inferior oblique muscle relative to the orbital implant and the point of insertion. RESULTS: In the enucleation patients, the inferior oblique muscle was anteriorly displaced and the muscle appeared to insert into an inferior subconjunctival scar mass in three of the four patients. In all four of the evisceration patients, the inferior oblique muscle appeared normally positioned and inserted onto the implant in the normal location. CONCLUSION: Enucleation without suturing of the inferior oblique muscle to the implant is associated with healing in an abnormal anterior location and into an inferior subconjunctival scar mass. Evisceration does not appear to disrupt the normal position or insertion of the inferior oblique muscle.

A sensitive and specific polymerase chain reaction-based assay for the diagnosis of cytomegalovirus retinitis.

PURPOSE: To develop a sensitive and specific laboratory assay for the diagnosis of cytomegalovirus retinitis. METHOD: We used a polymerase chain reaction-based assay for detection of cytomegalovirus DNA in vitreous samples. We attempted to detect cytomegalovirus DNA in 19 vitreous samples from patients with the acquired immunodeficiency syndrome (AIDS) who had untreated cytomegalovirus retinitis and in 40 vitreous samples from patients with AIDS who had been treated with systemic ganciclovir or foscarnet, or both. We also attempted to detect cytomegalovirus DNA in vitreous samples from 54 immunocompetent patients, including 32 with retinal detachment or macular hole, 11 with vitreous inflammation, and 11 with vitreous hemorrhage. Additionally, we attempted to detect cytomegalovirus DNA in 15 vitreous samples from patients with AIDS who had vitreoretinal inflammation not caused by cytomegalovirus. RESULTS: Cytomegalovirus DNA was detected in 18 of 19 eyes with untreated cytomegalovirus retinitis. We detected cytomegalovirus DNA in 19 of 40 vitreous samples from patients with previously treated cytomegalovirus retinitis. Cytomegalovirus DNA was not detected in any of 69 patients who did not have a clinical diagnosis of cytomegalovirus retinitis. Thus, the assay had an estimated sensitivity of 95% in detecting untreated cytomegalovirus retinitis and a sensitivity of 48% in detecting cytomegalovirus retinitis that had been treated with systemic ganciclovir or foscarnet, or both. The assay did not give false-positive results in patients with vitreous hemorrhage or vitreous inflammation. Most important, the assay did not give false-positive results in AIDS patients with vitreous inflammation from causes other than cytomegalovirus retinitis. CONCLUSION: We have developed a sensitive and specific diagnostic assay that will assist in the diagnosis of cytomegalovirus retinitis.

An orbital abscess secondary to acute dacryocystitis.

An orbital abscess is an ophthalmic surgical emergency that is typically caused by the spread of bacteria from adjacent structures, such as the sinuses, eyelids, or teeth. Although acute dacryocystitis is commonly associated with preseptal cellulitis, it rarely causes orbital infection. Infection of the lacrimal sac will typically localize in the preseptal space because the lacrimal sac lies anterior to the orbital septum. To the authors' knowledge, this is the first report of an intraconal abscess secondary to acute dacryocystitis. The key points in the surgical management of this entity are discussed.

Intracellular calcium regulates basolateral potassium channels in a chloride-secreting epithelium.

The two individual cell membranes of epithelia are functionally coupled, so that changes in apical membrane conductance are paralleled by changes in basolateral K+ conductance. However, the signal that regulates basolateral K+ conductance, thereby coupling the two membranes, is unknown. We tested the hypothesis that the cellular calcium concentration, [Ca2+]c, may regulate basolateral K+ conductance in canine tracheal epithelium, a Cl- -secreting epithelium that shows marked membrane coupling. Three findings support the hypothesis. First, the intracellular Ca2+ antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) attenuated the secretory response. Second, the secretagogue epinephrine increased [Ca2+]c, as measured with quin-2. Third, we found a K+ channel that was activated by Ca2+ on the cytosolic side of the membrane. Thus, cytosolic Ca2+ regulates the basolateral K+ conductance and may be the signal responsible for functional coupling of the two cell membranes.

Regulation of Cl- and K+ channels in airway epithelium.

Stimulation of transepithelial Cl- secretion by the airway epithelium requires activation of channels at the two opposite sides of the cell: the apical and basolateral membranes. At the apical membrane, the Cl- channel is regulated by phosphorylation with PKA and PKC. At the basolateral membrane, the KCLIC channel is regulated by [Ca2+]c. Addition of a secretagogue that increases cellular levels of cAMP also causes release of Ca2+ from intracellular stores. The Ca2+ may then regulate basolateral membrane KCLIC channels. The cAMP-induced increase in [Ca2+]c and activation of the KCLIC channel is transient, however, whereas activation of the Cl- channel and stimulation of secretion is a more sustained response. Those results suggest that the presence of a second Ca2(+)-independent K+ channel located at the basolateral membrane, which is only expressed in cells grown on permeable supports.

Calcium-activated potassium channels in canine airway smooth muscle.

Airway smooth muscle cells from canine trachealis muscle were dispersed by treatment with collagenase and elastase. Cells were identified as smooth muscle by their binding of anti-smooth muscle gamma-isoactin monoclonal antibodies and by their contraction in response to acetylcholine. The patch-clamp technique was used to study single channel currents in cell-attached and isolated patches of membrane. The most common single channel currents had a conductance of 266 +/- 12 pS (mean +/- S.D., n = 7) in symmetrical 135 mM-K solutions. The reversal potential of the channel was unaltered by large chemical gradients for Cl, Na and Ca and was determined exclusively by the chemical K gradient. Thus, the channel is highly selective for K. In both cell-attached and isolated patches of membrane, depolarization increased the frequency of channel opening and the duration of the open state. In isolated patches of membrane, increasing [Ca] on the cytoplasmic side of the membrane from 10(-8) to 10(-6) M increased both the frequency of channel opening and the duration of the open state. Tetraethylammonium, tetramethylammonium, or Cs (10 mM) on the cytoplasmic side of the membrane caused a voltage-dependent decrease in conductance of the open channel while having no obvious effect on channel kinetics. These blocks were completely reversible. Ba (10 mM) on the cytoplasmic side of the membrane slightly decreased inward currents and completely blocked outward currents through the channel. External Ba (10 mM) caused a voltage-dependent decrease in inward current. External tetraethylammonium (10 mM) completely blocked single channel currents.

Traumatic neuropathies of the optic nerve, optic chiasm, and ocular motor nerves.

This review focuses on traumatic chiasmal syndrome and traumatic neuropathies of cranial nerves II, III, IV, and VI. The review highlights common anatomical sites of injury to the above structures. Special emphasis is placed on review of recent literature. Other review of related material include.

Basolateral K+ channels in airway epithelia. II. Role in Cl- secretion and evidence for two types of K+ channel.

We previously described a Ca2(+)-activated K+ channel (KCLIC) in airway epithelial cells [J. D. McCann, J. Matsuda, M. Garcia, G. Kaczorowski, and M. J. Welsh. Am. J. Physiol 258 (Lung Cell. Mol. Physiol. 2): L334-L342, 1990]. To determine whether the KCLIC channel is a basolateral membrane channel and to understand its role in Cl- secretion, we studied airway epithelial cells grown on permeable supports. When cells were stimulated with A23187, charybdotoxin (ChTX) inhibited Cl- secretion and 86Rb efflux at the same concentrations, indicating that the KCLIC channel is required for Ca2(+)-stimulated Cl- secretion. We also investigated the function of K+ channels in adenosine 3',5'-cyclic monophosphate-stimulated secretion. Addition of isoproterenol caused a biphasic increase in Cl- secretion; the time course of the transient component correlated with the time course of the isoproterenol-induced increase in Ca2+ concentration [( Ca2+]c). ChTX inhibited the transient component, but not the prolonged component of secretion; Ba2+ inhibited the sustained component. These results suggest that when cells are grown on permeable supports isoproterenol-induced secretion depends on activation of two types of K+ channel: the KCLIC channel that is stimulated initially and a ChTX-insensitive K+ channel that is stimulated during sustained secretion. This conclusion was supported by measurement of 86Rb efflux from cell monolayers.