Sapecin B alters kinetic properties of rapidly inactivating K(+) channels in rat pituitary GH(3) cells.

Sapecin B is an antibacterial protein isolated and purified from culture medium of the embryonic cell line derived from the flesh fly (Sarcophaga peregrina). It has structural similarities to the scorpion toxin charybdotoxin (CTX). We have first detailed the effects of the newly described toxin (sapecin B) on the gating kinetics of the 4-aminopyridine-sensitive, rapidly inactivating K(+) current in rat pituitary GH(3) cells in order to investigate this protein's site of action, with whole-cell voltage-clamp methods. We have found that sapecin B alters the kinetics of activation and deactivation whereas there was no effect on the inactivation process. None of the effects of sapecin B was voltage dependent. In addition, sapecin B reduced whole-cell conductance. We suggest that the toxin may be ineffective against the voltage-sensitive segment, as well as the N-terminal and C-terminal domains, and CTX and sapecin B probably may have different binding sites.

Comparison of threshold visual perimetry and objective pupil perimetry in clinical patients.

OBJECTIVES: In an attempt to measure the visual field objectively, we have performed pupil perimetry, by which the pupil light reflex is monitored in response to perimetric light stimuli. The purpose of this study was to ascertain whether pupil perimetry reveals defects similar to those revealed by standard threshold perimetry in patients with various diseases. MATERIAL AND METHODS: An infrared pupillometer was linked to an automated perimeter to record, at each perimetric location, 76 pupil contractions, which were comparable to the test locations of the Humphrey Field Analyzer (HFA 30-2 program; Humphrey, San Leandro, CA). One hundred eighteen patients with various diseases were investigated. RESULTS: Ninety-one patients (77.1%) maintained a pupil area large enough (more than 10 mm2 in area) to respond adequately to focal light stimuli throughout the test. The correlation between the pupil field and the threshold visual field was subjectively judged to be good in most cases. However, pupil perimetry showed less damage than that seen in threshold perimetry in six of nine patients who had Leber's hereditary optic neuropathy (LHON). CONCLUSIONS: Pupil perimetry is a good method for measuring the visual field objectively and has potential for clinical use in most of the cases.

Augmentation of transient low-threshold Ca2+ current induced by GTP-binding protein signal transduction system in GH3 pituitary cells.

We characterized the effects of thyrotropin-releasing hormone (TRH; 500 nM) and guanosine 5'-0-3-thiotriphosphate (GTP gamma S; 50 microM) on two types of Ca2+ currents in pituitary-hormone-secretory GH3 cells and were surprised to find marked increases in transient, low-threshold Ca2+ currents (T currents) induced by extracellularly applied TRH or intracellularly applied GTP gamma S. The effect of TRH was blocked by intracellularly applied guanosine 5'-0-2-thiodiphosphate (GDP beta S; 100 microM). The increase in the T current was found to be accompanied by a decrease in long-lasting, high-threshold Ca2+ current (L-current), in response to both TRH or GTP gamma S. These indicate that the enhancement of Ca2+ influx by TRH (500 nM) is largely conferred by T currents in GH3 cells. A reduced concentration of TRH (5 nM) still markedly increased the T current, but failed to decrease the L current. These data suggest that the augmentation of the T currents as well as depression of the L currents by TRH (500 nM), through the activation of a GTP-binding protein, may constitute an important regulatory mechanism of sustained pituitary hormone secretion in GH3 cells.

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells.

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.

A computer-based model of parasystolic activity modulated by ventricular activation in the open-chest dog.

The modulated parasystole hypothesis proposed by Moe has been suggested as a common mechanism of a wide variety of arrhythmias. To represent the arrhythmic patterns defined by the model as an actual ECG trace, a "hybrid" experimental system was devised. The system consisted of the intact canine heart connected to a microcomputer that operated as the modulated parasystolic pacemaker. An intrinsic cycle length of the "ectopic" pacemaker was modulated by the phase-dependent effect of the activity of the intact heart in situ. Discharges in the "ectopic" pacemaker were transmitted to the right ventricle. The result was that the theoretically defined patterns of the ectopic activity were represented as real ECG traces. Some published examples of intermittent parasystole that had been attributed to other complex mechanisms were accurately reproduced when the estimated phase response curves were set in the "ectopic" pacemaker. A wide variety of clinical eCG tracings of ventricular arrhythmias may be reproduced by this hybrid experimental model.

A method for tracking the locomotion of an isolated microorganism in real time.

An inexpensive, real-time recording system ("bug-tracker") for tracking the movements of an isolated microorganism was assembled using a close-up video system, a video memory card, and a personal computer. An isolated organism moving in an almost two-dimensional plane is viewed by the close-up video camera, and a selected video frame is digitized by the video memory card into 256 x 256 pixels (picture points). The pixels of the ith frame are subtracted from those of the i-lth frame to delete images other than that of the image of the moving organism. Windows with an optimized size are generated commonly in the ith and i-lth frame to reduce the number of pixels directly accessed by the computer, and the address of the pixel with the largest value inside the windows identifies the coordinates of the organism on the X and Y axes. By optimizing the size of a window for a given organism, the sampling for the X and Y coordinates can be made at times separated by approximately 0.2 to 2.0 seconds. Data are automatically filed on a floppy disk.

The Paramecium circadian behavioral rhythm: light phase response curves and entrainment.

The population of a ciliate protozoan, Paramecium multimicronucleatum, exhibits a circadian rhythm as measured by the number of the cells traversing an observation point ("traverse frequency," or TF). The present study examined phase shifting of the TF rhythm by administering 2-hr light pulses at different phases of the circadian cycle to cultures free-running in constant darkness (DD). The results were summarized in a phase response curve (PRC), categorized as Type 1. This PRC indicated a relatively narrow phase zone insensitive to the light pulse ("dead zone"). Entrainment of the rhythm to light pulses repeated at 24-hr intervals was also examined, and it was found that the rhythm gradually reached a steady state, following several transient cycles, with the pulses falling at a phase corresponding to the narrow dead zone. Such a steady-state rhythm, with a minimum at approximately 3 hr after the pulse and a maximum at approximately 12 hr after the pulse, was mathematically simulated by superimposing a response function to the pulse on a sinusoidal function representative of the free-running rhythm in DD.

Microcomputerized measurement of the circadian locomotor rhythm in microorganisms.

A computerized close-up video/photoamplifier system was implemented for the study of circadian locomotor rhythm in a population of a ciliate protozoan, Paramecium. This fully microcomputerized system facilitated automatic long-term measurement of three parameters in parallel: (1) numbers of specimens per 10 min traversing beneath a fixed point in an experimental vessel, (2) times taken by specimens to traverse the point, and (3) interval times between subsequent specimens traversing the point. Stochastic analyses using these parameters can derive the circadian fluctuation of physiological variables, such as swimming speed and the frequency of avoiding reaction (abrupt change in swimming direction). The computerized system simultaneously accomplished the acquisition of these three sets of data, their transient storage, and their graphic display according to given format. The system software was constituted so that an experimenter with little computer knowledge, could easily operate the system by answering questions displayed on the computer monitor.