Cochlear implantation in auditory neuropathy spectrum disorders: role of transtympanic electrically evoked auditory brainstem responses and serial neural response telemetry.

OBJECTIVE: To evaluate the utility of pre-operative transtympanic electrically evoked auditory brainstem responses and post-operative neural response telemetry in auditory neuropathy spectrum disorder patients. METHODS: Four auditory neuropathy spectrum disorder patients who had undergone cochlear implantation and used it for more than one year were studied. All four patients underwent pre-operative transtympanic electrically evoked auditory brainstem response testing, intra-operative and post-operative (at 3, 6 and 12 months after switch-on) neural response telemetry, and out-patient cochlear implant electrically evoked auditory brainstem response testing (at 12 months). RESULTS: Patients with better waveforms on transtympanic electrically evoked auditory brainstem response testing showed superior performance after one year of implant use. Neural response telemetry and electrically evoked auditory brainstem response measures improved in all patients. CONCLUSION: Inferences related to cochlear implantation outcomes can be based on the waveform of transtympanic electrically evoked auditory brainstem responses. Robust transtympanic electrically evoked auditory brainstem responses suggest better performance. Improvements in electrically evoked auditory brainstem responses and neural response telemetry over time indicate that electrical stimulation is favourable in auditory neuropathy spectrum disorder patients. These measures provide an objective way to monitor changes and progress in auditory pathways following cochlear implantation.

Glucagon-like peptide-1 stimulates insulin secretion by a Ca2+-independent mechanism in Zucker diabetic fatty rat islets of Langerhans.

This study investigates the mechanisms responsible for glucagon-like peptide-1 (GLP-1)-induced insulin secretion in Zucker diabetic fatty (ZDF) rats and their lean control (ZLC) littermates. Glucose, and 100 nmol/L GLP-1 (7-37 hydroxide) in the presence of stimulatory glucose concentrations, induced insulin secretion in islets from ZLC animals. In contrast, ZDF islets hypersecreted insulin at low glucose (5 mmol/L) and were poorly responsive to 15 mmol/L glucose stimulation, but increased insulin secretion following exposure to GLP-1. The insulin secretory response to 100 nmol/L GLP-1 was reduced by 88% in ZLC islets exposed to exendin 9-39. The intracellular Ca2+ concentration ([Ca2+]i) increased in fura-2-loaded ZLC islets following stimulation with 12 mmol/L glucose alone or GLP-1 in the presence of 12 mmol/L glucose. The increases in [Ca2+]i and insulin secretion in ZLC islets induced by GLP-1 were attenuated by 1 micromol/L nitrendipine. In contrast, neither glucose nor GLP-1 substantially increased [Ca2+]i in ZDF islets. Furthermore, insulin secretory responses to GLP-1 were not significantly inhibited in ZDF islets by nitrendipine. However, the insulin secretory response to GLP-1 in both ZLC and ZDF islets was ablated by cholera toxin. Our findings indicate that in ZLC islets, GLP-1 induces insulin secretion by a mechanism that depends on Ca2+ influx through voltage-dependent Ca2+ channels, whereas in ZDF islets, the action of GLP-1 is mediated by Ca2+-independent signaling pathways.

Characterization of a Ca2+ release-activated nonselective cation current regulating membrane potential and [Ca2+]i oscillations in transgenically derived beta-cells.

Although stimulation of insulin secretion by glucose is regulated by coupled oscillations of membrane potential and intracellular Ca2+ ([Ca2+]i), the membrane events regulating these oscillations are incompletely understood. In the presence of glucose and tetraethylammonium, transgenically derived beta-cells (betaTC3-neo) exhibit coupled voltage and [Ca2+]i oscillations strikingly similar to those observed in normal islets in response to glucose. Using these cells as a model system, we investigated the membrane conductance underlying these oscillations. Alterations in delayed rectifier or Ca2+-activated K+ channels were excluded as a source of the conductance oscillations, as they are completely blocked by tetraethylammonium. ATP-sensitive K+ channels were also excluded, since the ATP-sensitive K+ channel blocker tolbutamide substituted for glucose in inducing [Ca2+]i oscillations. Thapsigargin, which depletes intracellular Ca2+ stores, and maitotoxin, an activator of nonselective cation channels, both converted the glucose-dependent [Ca2+]i oscillations into a sustained elevation. On the other hand, both SKF 96365, a blocker of Ca2+ store-operated channels, and external Na+ removal suppressed the glucose-stimulated [Ca2+]i oscillations. Maitotoxin activated a nonselective cation current in betaTC3 cells that was attenuated by removal of extracellular Na+ and by SKF 96365, in the same manner to a current activated in mouse beta-cells following depletion of intracellular Ca2+ stores. Currents similar to these are produced by the mammalian trp-related channels, a gene family that includes Ca2+ store-operated channels and inositol 1,4,5-trisphosphate-activated channels. We found several of the trp family genes were expressed in betaTC3 cells by reverse transcriptase polymerase chain reaction using specific primers, but by Northern blot analysis, mtrp-4 was the predominant message expressed. We conclude that a conductance underlying glucose-stimulated oscillations in beta-cells is provided by a Ca2+ store depletion-activated nonselective cation current, which is plausibly encoded by homologs of trp genes.

Expression and function of pancreatic beta-cell delayed rectifier K+ channels. Role in stimulus-secretion coupling.

Voltage-dependent delayed rectifier K+ channels regulate aspects of both stimulus-secretion and excitation-contraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (betaTC3-neo) and beta-cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion. Using reverse-transcription polymerase chain reaction methods to amplify all known candidate delayed rectifier transcripts, the expression of the K+ channel gene Kv2.1 in betaTC3-neo insulinoma cells and purified rodent pancreatic beta-cells was detected and confirmed by immunoblotting in the insulinoma cells. betaTC3-neo cells were also found to express a related K+ channel, Kv3.2. Whole-cell patch clamp demonstrated the presence of delayed rectifier K+ currents inhibited by tetraethylammonium (TEA) and 4-aminopyridine, with similar Kd values to that of Kv2.1, correlating delayed rectifier gene expression with the K+ currents. The effect of these blockers on intracellular Ca2+ concentration ([Ca2+]i) was studied with fura-2 microspectrofluorimetry and imaging techniques. In the absence of glucose, exposure to TEA (1-20 mM) had minimal effects on betaTC3-neo or rodent islet [Ca2+]i, but in the presence of glucose, TEA activated large amplitude [Ca2+]i oscillations. In the insulinoma cells the TEA-induced [Ca2+]i oscillations were driven by synchronous oscillations in membrane potential, resulting in a 4-fold potentiation of insulin secretion. Activation of specific delayed rectifier K+ channels can therefore suppress stimulus-secretion coupling by damping oscillations in membrane potential and [Ca2+]i and thereby regulate secretion. These studies implicate previously uncharacterized beta-cell delayed rectifier K+ channels in the regulation of membrane repolarization, [Ca2+]i, and insulin secretion.

Immunocytochemical detection of extracellular annexin II in cultured human skin keratinocytes and isolation of annexin II isoforms enriched in the extracellular pool.

Monoclonal antibodies were raised against trypsinized human skin epidermal cells and selected for their staining of the epidermal cells in a cell periphery pattern. One antibody, CP-1, immunoprecipitated a 36 kDa protein that was identified as annexin II heavy chain by microsequencing of a CNBr-generated peptide fragment from the antigen and by cross-identification with another anti-annexin II antibody. In addition to staining a broad cell periphery band in keratinocytes, CP-1 also detected annexin II outside and in between the top layer cells before cell permeabilization. Double-labeling of annexin II and F-actin revealed a distinct topographical relationship between the two, with intercellular annexin II flanked by the submembranously located actin of the juxta-positioned cells. Annexin II was isolated from cultured keratinocytes via immunoaffinity column chromatography in one step, using the same monoclonal antibody CP-1 and was found to be resolved into multiple isoforms when analyzed by two-dimensional gel electrophoresis. The predominant components of annexin II were basic, with pI of 6.5-8.5, and some of them formed disulfide-linked monomeric multimers under non-reducing conditions. Acidic annexin II isoforms with pI 5.4-5.8 were barely detectable among the total annexin II isolated but were selectively enriched in an extracellular pool created by 0.05% ethylenediaminetetraacetic acid (EDTA) dispersion of the cultured cells into single cell suspensions. Furthermore, they can be separated from the rest of annexin II by using a different elution condition. A 46 kDa protein, the identity of which is unclear, co-eluted with the acidic isoforms in the EDTA washes. These acidic isoforms, which co-eluted with the 46 kDa protein, are suspected of corresponding to the extracellular annexin II detected immunocytochemically.