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Biomedical subjects

M Sears

Publications and source records attributed to M Sears.

At least 19 recordsLinked to original sources
PubMed2001

Regular versus as-needed short-acting inhaled beta-agonist therapy for chronic obstructive pulmonary disease.

Regular short-acting inhaled beta-agonist therapy is of uncertain benefit in patients with chronic obstructive pulmonary disease (COPD). We conducted a randomized, concealed, double-blind, placebo-controlled crossover trial in two periods, each of 3-mo duration, involving 53 patients with a smoking history of > 20 pack-years, an FEV1 of < 70% predicted, and an FEV1/VC ratio of < 0.7 after inhalation of 200 microg albuterol. All patients received regular ipratropium bromide at 20 microg per puff in 2 puffs four times daily, beclomethasone at 250 microg per puff or equivalent corticosteroid in 2 puffs twice daily, and open-label inhaled albuterol as needed. Interventional therapy consisted of regular inhaled albuterol (100 microg per puff, in 2 puffs four times daily) versus placebo. Patients used twice as much active albuterol in the regular use period (mean: 8.07 puffs of coded and 4.68 puffs of open-label medication; total: 12.75 puffs daily) than during the as-needed period (mean: 6.34 puffs of open-label albuterol daily). Despite greater beta-agonist use, patients showed similar results during treatment and control periods for all outcomes. Differences between active and placebo periods were: FEV1: -0.04 L (95% confidence interval [CI]: -0.09 to 0.01 L); slow vital capacity: 0.04 L (95% CI: -0.12 to 0.20 L); 6-min walk test distance: -3.1 m (95% CI: -16.8 to 10.5 m); and Chronic Respiratory Questionnaire scores for dyspnea: 0.02 (95% CI: -0.13 to 0.16); fatigue: -0.02 (95% CI: -0.25 to 0.20); mastery: 0.01 (95% CI: -0.20 to 0.24); and emotional function: 0.02 (95% CI: -0.20 to 0.24). We found that in patients with COPD, use of regular short-acting inhaled beta-agonists resulted in twice as much beta-agonist use without physiologic or clinical benefit as did use on an as-needed basis.

Administration, Inhalation↗
PubMed1998

pICln can regulate swelling-induced Cl- currents in either layer of rabbit ciliary epithelium.

Swelling-induced Cl- currents were investigated in freshly prepared non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells of the rabbit ciliary body using the whole-cell patch clamp technique. Exposure of both NPE and PE cells to hypotonic stress induced Cl- currents that exhibited outward rectification and were insensitive to Ca+2. We found that swelling-induced Cl- currents in PE cell are observed shortly after isolation. The swelling-induced Cl- current showed little or no inactivation at positive membrane voltages and was sensitive to 100 microM NPPB and 100 microM DIDS. Injection of cRNA encoded rabbit pICln into Xenopus oocytes produced an outwardly rectifying Cl- current displaying features consistent with the swelling-induced Cl- current in epithelium. pICln is ubiquitous in the ciliary epithelium. It participates in the equilibration of short term tonicity alterations, a phenomenon underlying mechanisms with larger and slower amplitudes for aqueous secretion by these cells.

Animals↗
PubMed1998

Adrenergic-mediated connexin43 phosphorylation in the ocular ciliary epithelium.

PURPOSE: Gap junctions provide metabolic cooperativity between the nonpigmented and pigmented cells of the ciliary epithelium. Connexin43 is the major protein of these junctions. To learn whether the phosphorylation state of this gap junction is sensitive to adrenergic mediators, we exposed isolated intact ciliary epithelia to agonists of the G-protein receptor-coupled system and analyzed the phosphorylation state of connexin43 by western blot. METHODS: The double layer of intact ciliary epithelia was isolated and exposed to isoproterenol, forskolin, and the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). The phosphorylation state of connexin43 was analyzed by western blot, using a monoclonal antibody that recognized both the phosphorylated and dephosphorylated connexin43. An upward shift in electrophoretic mobility confirmed the presence of a phosphate group. RESULTS: Connexin43 phosphorylation was rapidly induced by each of these agonists. One microM isoproterenol and 5 microM forskolin induced phosphorylation of connexin43, as did 16 nanomolar TPA. The effect of isoproterenol was partially blocked by 1 microM timolol. Addition of a phosphatase after forskolin treatment reversed the effect of forskolin. Control explant tissue not treated with these agents exhibited a slower but definite phosphorylation of connexin43. CONCLUSIONS: Phosphorylation of ciliary epithelial connexin43 is sensitive to modulators of the cAMP system as well as an agent that activates PKC. Isolated intact ciliary epithelia phosphorylate connexin43 by endogenous mechanisms, most likely as a protective response to stress.

Adrenergic beta-Agonists↗
PubMed1997

Cloning and functional expression of a swelling-induced chloride conductance regulatory protein, plCln, from rabbit ocular ciliary epithelium.

The cDNA encoding a swelling-induced chloride conductance regulatory protein, plcln, was cloned from rabbit ciliary epithelium by using a polymerase chain reaction (PCR)-based approach. The open reading frame encoding 236 amino acids possesses high amino acid identity (93/%) with the previously cloned plcln from human ciliary epithelium. Outwardly rectifying currents were recorded in Xenopus oocytes injected with plcln cRNA, a result consistent with plcln expression in ciliary epithelium. A widespread distribution and marked expression of plcln mRNA in both nonpigmented ciliary epithelial (NPE) cells and pigmented ciliary epithelial (PE) cells was found for the first time. In situ hybridization analysis showed that plcln expression is more abundant in NPE than PE. These findings are consistent with the idea that plcln may be an important regulatory element in these secretory cells.

Amino Acid Sequence↗
PubMed1997

Circadian aqueous flow mediated by beta-arrestin induced homologous desensitization.

It was hypothesized that homologous desensitization regulates signal transduction from the beta-adrenergic receptor in the ocular ciliary epithelium to affect the circadian rhythm of aqueous humor secretion. beta-arrestin-1 was cloned from the rabbit ciliary epithelium, and the full length cDNA used as a probe for Northern blot analysis to examine the diurnal expression of beta-arrestin mRNA. Protein expression of beta-arrestin-1 at intervals during the circadian cycle of aqueous secretion showed a decrease in beta-arrestin expression when maximal activation of the beta-adrenergic receptor is known to increase secretion. Diurnal expression of beta-arrestin suggests that homologous desensitization can regulate the circadian rhythm of aqueous flow.

Amino Acid Sequence↗
PubMed1997

Calcium signals from intact rabbit ciliary epithelium observed with confocal microscopy.

PURPOSE: We investigated patterns of evoked calcium signals to learn about the function of the calcium second messenger system in ciliary epithelium. METHODS: Isolated infact ciliary processes were loaded with fluo-3/AM and observed with a Bio-Rad MRC-600 laser scanning confocal imaging system, before, during, and after perfusion with catecholamines, cholinergic agents, and autocoids. RESULTS: One microM acetylcholine (ACH) and 10 microM carbachol (CARB) induced an atropine-sensitive increase in intracellular free calcium ion concentration ([Ca2+]i), considerably greater in NPE than in PE. 10 microM epinephrine (EPI) and 100 microM phenylephrine (PHE) increased [Ca2+]i in NPE and PE, in this case PE > NPE. These effects were blocked by prazosin. 10 mM caffeine (CAF) increased of [Ca2+]i in NPE and PE (NPE > PE) and sometimes produced very slow oscillations with an interval of 10 to 25 s. Prior administration of CAF strongly suppressed the effects of ACH, CARB, EPI, PHE, histamine, and adenosine 5-triphosphate (ATP). One hundred microM ryanodine (RYA) or thapsigargin (TG) increased [Ca2+]i in NPE and PE (NPE > PE). CONCLUSIONS: In the ciliary epithelium: (1) different patterns of evoked transients and oscillations of calcium were seen in response to agonists; (2) NPE appeared to contain a predominance of muscarinic receptors, while the PE is dominated by alpha 1-adrenergic receptors and (3) the increase in [Ca2+]i by CAF or RYA or TG in either cell layer and the blocking effects of these agents upon agonist, evoking increases in [Ca2+]i, suggested involvement of both the cyclic adenosine diphosphate ribose and the inositol 1, 4, 5 triphosphate (InsP3) systems in the regulation of intracellular calcium.

Aniline Compounds↗
PubMed1997

A low conductance chloride channel in the basolateral membranes of the non-pigmented ciliary epithelium of the rabbit eye.

PURPOSE: Chloride efflux plays an important role in aqueous humor production. Chloride currents have been described in bovine non-pigmented ciliary epithelium (NPE), in transformed cultured human NPE, and in bovine volume-activated chloride channels described in the latter. It is the basolateral membranes of the NPE of ocular ciliary processes that comprise the exit pathway for the process of aqueous secretion performed by the double syncytial layer of ciliary epithelium, however. Therefore, we studied both cell-attached, and, excised, inside-out patches from basolateral membranes of the NPE. METHODS: Cell-attached and cell-free excised patches were formed from the basolateral membranes of NPE and single channel currents recorded with a Dagan 3900A patch clamp amplifier. RESULTS: A low conductance channel of 14 pS was observed and recorded in 30% of cell attached patches and in 35% of excised inside out patches under symmetrical conditions (160 mM chloride). This channel displayed a nearly linear current-voltage relationship, with an open probability that was not voltage-dependent. The channel was chloride-selective: N-methyl-D-glucamine (NMDG) used as cation did not alter the current profile nor the reversal protein. Further, with inside-out patches, the reversal potential was close to zero (0.3 +/- 0.5 mV (10) in symmetrical (160 mV) chloride, but shifted to -32.3 +/- 0.5 mV (5) when the concentration of chloride in the bathing solution was reduced to 40 mM while the recording pipette was held at 160 mM. This value approaches the theoretical equilibrium potential of chloride for these conditions. The channel anion permeability sequence was: I- > NO3- > or = Br- > Cl- > gluconate- approximately equal to aspartate-. Three different chloride-channel blockers inhibited the channel activity. CONCLUSION: A low conductance channel, selective for chloride, and, modulated by beta adrenergic and VIP stimulation, based on it sensitivity to exogenously added cAMP, ATP and the catalytic subunit of PKA, is present in the exit basolateral membranes of rabbit NPE and contributes to the process of aqueous humor formation.

Adenosine Triphosphate↗
PubMed1996

Spontaneous and evoked oscillations of cytosolic calcium in the freshly prepared ciliary epithelial bilayer of the rabbit eye.

Under confocal microscopy, calcium imaging of the isolated secretory ciliary epithelium, especially the inner layer, non-pigmented epithelium (NPE), revealed spontaneous calcium oscillations in multiple patterns. Oscillations induced by stimulation of the muscarinic receptor have an onset that coincides with synthesis and release of InsP3. Caffeine (5 mM) induced a calcium transient and blocked spontaneous and (0.1 mM) muscarine induced oscillations. Thapsigargin (1 microM) prevented a muscarinic response. Muscarine and caffeine induced transients have significantly greater amplitude in NPE than in PE (outer layer, pigmented epithelium). Calcium transients and frequencies of spontaneous oscillations are greater in NPE than PE. In NPE muscarine induced oscillations are observed with higher frequencies than the spontaneous ones: gamma-0.24 +/- 0.05Hz (8) versus 0.08 +/- 0.01Hz (18). These repetitive responses, spontaneous and receptor coupled, reflect intracellular coding of information. A calcium signal may contribute to the regulation of aqueous humor formation.

Animals↗
PubMed1996

Postmortem diagnosis of Fusarium panophthalmitis by the polymerase chain reaction.

PURPOSE: We undertook this study to determine if a polymerase chain reaction-based test that we developed for the filamentary fungus, Fusarium, could be used to detect the organism in postmortem ocular tissues. METHODS: We applied the polymerase chain reaction to amplify a target fragment of Fusarium DNA from formalin-fixed ocular tissues from a patient with endogenous Fusarium panophthalmitis. RESULTS: By using the polymerase chain reaction-based test, we were able to amplify the target fragment of DNA from the infected eyes, but not from uninfected control eyes. CONCLUSIONS: The technique appears to hold promise to be a sensitive, specific, and rapid method of diagnosing Fusarium infections.

Blotting, Southern↗
PubMed1996

Transepithelial transport of ascorbic acid by the isolated intact ciliary epithelial bilayer of the rabbit eye.

The purpose of these experiments was to test whether the isolated intact epithelial bilayer derived from rabbit ciliary processes transports ascorbic acid and to what degree the transport rate in vitro corresponds to the in vivo process. The intact ciliary epithelial bilayer of the rabbit eye isolated by perfusion was mounted in a particularly constructed Ussing type chamber. Fluxes were measured by additions of 14C ascorbate to the hemichamber on either the pigmented epithelium (PE) or the nonpigmented epithelium (NPE) side where equal concentrations of ascorbate from .02 to 2.0 mM were present. Samples were taken at intervals thereafter and counted in a liquid scintillation counter. The experiments were done under short circuit conditions to avoid the possible influence of fluctuating currents upon the movement of ascorbate. Like the earlier iris ciliary body preparations, separation of ascorbate fluxes is also done by the isolated intact ciliary epithelial bilayer, and the transport of ascorbic acid proceeds by saturation kinetics. The uptake process is accomplished entirely by the pigmented epithelium (PE). The Km of the process is 0.97 mM, and Vmax was valued at 130 nM/L/hr. Thus, assuming an aqueous flow rate of 2 microliters/min, the transfer of ascorbic acid across the bilayer occurs at a rate required to maintain the ordinary millimolar concentration of ascorbic acid in the aqueous humor found in vivo.

Animals↗
PubMed1996

Regulation and bioelectrical effects of cyclic adenosine monophosphate production in the ciliary epithelial bilayer.

PURPOSE: To determine whether the intact isolated ciliary epithelial bilayer retains the alpha-2 and beta adrenergic receptor activation and interaction described for whole ciliary processes and whether this pure epithelial bilayer displays bioelectric parameters sensitive to alterations in cyclic adenosine Monophosphate (cAMP) production induced by adrenergic compounds. METHODS: The intact ciliary epithelial bilayer of the rabbit eye isolated by perfusion was mounted in a specially constructed Ussing-type chamber. The transepithelial potential difference and short-circuit current were monitored for effects induced by agents that stimulated or blocked (some did both) caMP production. Using a radioimmunoassay, the latter were studied in the pure epithelial bilayers and in whole ciliary processes. RESULTS: A reproducible increase in cAMP production and an increase in the short-circuit current induced in the bilayer by isoproterenol, a nonspecific beta adrenergic agonist, were both blocked by pretreatment with either timolol, a nonspecific beta adrenergic blocking agent, or with para-aminoclonidine, an alpha-2 agonist. Maximal stimulation of cAMP with forskolin in this pure isolated epithelial preparation yields a response that is 60% of the value found in whole processes, indicating that the latter tissue contains responsive sites that are nonepithelial, probably vascular, or perhaps stromal. The degree of inhibition of the beta adrenergic receptor by alpha-2 agonists was not very different in the two preparations. On the other hand, inhibition of the epithelial vasointestinal peptide receptor by neuropeptide Y or alpha-2 agonism was considerably heightened in the pure bilayered epithelial preparation. CONCLUSIONS: The isolated intact ciliary epithelial bilayer, when stimulated with beta adrenergic receptor agonists, vasointestinal peptide, or forskolin, produces increased cAMP and its transepithelial potential becomes hyperpolarized. These chemical and bioelectrical effects are prevented by pretreatment with either alpha-2 adrenergic agonists or beta adrenergic blocking agents. The results obtained in the isolated intact purely epithelial ciliary bilayer confirm that the ciliary epithelium is the source of adrenergic receptor activation and interaction and support the hypothesis that aqueous humor production is regulated by interactions between epithelial alpha-2 and beta adrenergic receptors.

Adrenergic alpha-Agonists↗
PubMed1990

Metabolic neuropathy.

In this article a review and summary of peripheral neuropathies secondary to metabolic disorders, affecting the lower extremity, have been presented. The clinical presentation of individual symptomatology is variable depending on the nature and chronicity of illness. The clinical features that the podiatric physician must recognize is weakness, sensory, and gait disturbances resulting from these syndromes. Some disorders are reversible if recognized and treated early in their course. Thus, a thorough general history and physical examination is required of all patients presenting with apparent podiatric symptomatology.

Diagnosis, Differential↗
PubMed1989

Adenylate cyclase stimulation and intraocular pressure reduction by forskolin analogs.

Forskolin and 18 chemical analogs of forskolin were assayed for stimulation of adenylate cyclase activity in vitro and for their effects on intraocular pressure (IOP) in vivo. Adenylate cyclase activity was determined with a preparation of corpora striata from male Wistar rats and test drug concentrations of 0.3 to 300 microM. IOP effect was monitored after a single topical ocular application of a 1% suspension of the test drug to male New Zealand albino rabbits. Significant reductions in IOP occurred with compounds which had potent cyclase stimulatory properties. Most compounds with little cyclase stimulatory effect produced little or no decrease in IOP. A 1-substituted morpholino-acetoxy derivative, which may be a prodrug metabolized to active form by corneal esterases, showed little cyclase stimulation in vitro but produced significant reductions in IOP.

Adenylyl Cyclases↗