Effects of thapsigargin and cyclopiazonic acid on twitch force and sarcoplasmic reticulum Ca2+ content of rabbit ventricular muscle.

Thapsigargin (TG) and cyclopiazonic acid (CPA) are reported to be specific high-affinity inhibitors of the sarcoplasmic reticulum (SR) Ca2+ pump in isolated membranes and cells, with TG causing complete pump inhibition at nanomolar concentrations. To evaluate the effectiveness of TG and CPA in small multicellular cardiac preparations, we used rapid cooling contractures (RCCs) to assess the SR Ca2+ load. In contrast to observations in single myocytes, TG caused remarkably slow and incomplete SR Ca2+ depletion in multicellular preparations. A 45-minute exposure to 500 microM TG at 30 degrees C and 0.5-Hz stimulation only decreased RCCs by 76 +/- 5% (and 100 microM CPA reduced RCCs by 59 +/- 10% [mean +/- SEM]). In contrast, 10 minutes with 20 mM caffeine completely abolished RCCs. This confirms that there was still a caffeine-sensitive pool of Ca2+ in the TG-treated muscle. The time constant of rest decay of RCCs was accelerated by both TG (from 83 +/- 18 to 26 +/- 6 seconds) and CPA (from 68 +/- 11 to 10 +/- 5 seconds). This might be expected since Ca2+ leaking from the SR during rest cannot be taken back up as efficiently, favoring Ca2+ extrusion by the sarcolemmal Na(+)-Ca2+ exchanger. TG and CPA decreased twitch force (by 44 +/- 7% and 40 +/- 11%, respectively) and increased twitch duration, presumably because of the SR effects. We conclude that complete blockade of SR Ca2+ uptake by TG or CPA in multicellular preparations cannot be assumed, even at high [TG] or [CPA], unless evaluated (eg, by RCC).

Ciliary beat frequency of hamster oviducts is decreased in vitro by exposure to solutions of mainstream and sidestream cigarette smoke.

Epidemiological data support a correlation between smoking and increased incidence of ectopic pregnancy, yet the causal mechanism responsible for this relationship is unknown. The purpose of this study was to examine the effect of solutions containing dissolved mainstream (MS) or sidestream (SS) cigarette smoke on ciliary beat frequencies (CBF) in explants of hamster oviducts. MS smoke is the puff inhaled by an active smoker, while SS smoke leaves the burning end of cigarette. SS smoke is inhaled by both active and passive smokers. Experiments were performed in handmade perfusion chambers using infundibula from hamster oviducts. After a short incubation in Earle's balanced salt solution containing HEPES buffer (EBSS-H), chambers were flushed with one of six types of smoke solution prepared in EBSS-H, and incubation continued 19 min. A second perfusion (washout) was then done using EBSS-H alone to determine whether effects induced by the smoke solutions could be reversed. CBF were determined at three times in both the smoke and washout solutions, and means were compared to values obtained in the initial EBSS-H incubation. All smoke solutions except the SS particulate solution inhibited CBF in a dose-dependent manner. Whole MS and whole SS smoke solution at the highest strength tested caused the greatest inhibition and in some cases completely stopped ciliary beating. Both single-strength and 0.1-strength MS gas phase solutions, which contained concentrations of nicotine in the range found in typical human smokers, produced about 50% inhibition of ciliary beating. Inhibition was generally seen within 2-12 min of adding smoke solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of ciliary beat frequencies in hamster oviducal explants.

We have developed a simple direct method, requiring minimal manipulation, to measure beat frequencies of the cilia on the external surface of hamster oviducal infundibula in vitro. Two perfusion chambers (closed and open) were used; both can be hand-made in a few minutes and discarded after use. Ciliary beat frequencies were determined by measuring variations in light intensity with time in a single pixel positioned over a video image of the beating cilia. Data files were collected using Image 1 software and later transferred to PSI Plot or Lotus 123 spreadsheets for analysis by counting the number of brightness peaks recorded per second or by subjecting the data to Fourier transformation with or without smoothing. These methods of analysis gave similar results. To verify that Image 1 data files contain accurate representations of CBF, videotapes of beating cilia were made and subjected to frame-by-frame analysis. Image 1 interfaced with a standard video camera was found to collect reliable data over a beat frequency range of 0-15 cycles/sec. In some Fourier transforms, secondary peaks were observed and were shown to represent cilia beating at more than one frequency in a sampled region. Coefficients of variation for repeated measurements taken on the same region varied from 4.1% to 9.0%. Small but significant differences were found between beat frequencies at different regions of the same oviduct. When chambers were perfused discontinuously and measurements of beat frequency were made at least 5 min after each perfusion, no effect of perfusion on frequencies was observed. However, during continual perfusion of the open chamber, a slight but significant increase in beat frequency was observed after perfusion was initiated. Muscle contraction, which sometimes occurs in the open chamber, did not affect beat frequency measurements. Infundibula could be stored at 4 degrees C overnight without any negative effect on beat frequencies. Cold storage also reduced muscle contraction. Placement of a small coverslip on infundibula in the open chambers was also found to reduce muscle contraction and facilitate beat frequency measurements. Coverslipping did not affect beat frequencies. This method of beat frequency analysis will be valuable for analyzing factors that regulate or influence cilia in mammalian oviducts.