Synchronization between beating cilia.

A novel quantitative parameter is proposed to define and measure the degree of synchronization between two small ciliary areas. These areas can be close to or far from one another. The Pearson correlation factor is used to define the degree of synchronization by a single number. This approach is based on a computerized, dual photoelectric method which simulataneously measures the scattered light from two small areas on the ciliary epithelium or its tissue culture. The measurements were performed on tissue culture from frog's palate epithelium. It was found that: (a) the degree of synchronization decreases, as a function of distance; (b) the correlation is fairly high even at relatively large separations, when measured on the same patch; (c) on a given patch the synchronization factor is independent of the direction of the metachronal wave; (d) close disconnected ciliary cells exhibit fairly high correlation; (e) disconnected randomly choosen ciliary cells at relatively large separation distances exhibit relatively low correlation, smaller by a factor of 2 than the correlation factor at the same distances when measured along the metachronal wave; (f) the average frequencies' ratio and the metachronal wavelength can be used as first-order indicators of ciliary synchronization; (g) there is a spread of metachronal wavelengths even over a single well-organized patch.     

Outward sodium current in beating heart cells.

This article is a study of the fast Na current during action potentials. We have investigated the outward Na current (Mazzanti, M., and L.J. DeFelice. 1987. Biophys. J. 52:95-100) in more detail, and we have asked whether it goes through the same channels associated with the rapid depolarization phase of action potentials. We address the question by patch clamping single, spontaneously beating, embryonic chick ventricle cells, using two electrodes to record the action potential and the patch current simultaneously. The chief limitation is the capacitive current, and in this article we describe a new method to subtract it. Varying the potential and the Na concentration in the patch pipette, and fitting the corrected currents to a standard model (Ebihara, L., and E.A. Johnson. 1980. Biophys. J. 32:779-790), provides evidence that the outward current is carried by the same channels that conduct the inward current. We compare the currents in beating cells to currents in nonbeating cells using whole-cell and cell-attached patch clamp recordings. The latter tend to show more positive Na reversal potentials, with the implication that internal Na is higher in beating cells. We propose that the plateau of the action potential, which is partly due to an inward Ca current, exceeds Na action current reversal potentials, and that this driving force gives rise to an outward movement of Na ions. The existence of such a current would imply that the fast repolarization phase after the upstroke of cardiac action potentials is partly due to the Na action current.     

Simultaneous measurement of ciliary beating and intracellular calcium.

A novel system for measuring, simultaneously, ciliary beating and intracellular free calcium is presented. The advantages and dynamic nature of the system are demonstrated by measuring the effects of the calcium ionophore lonomycin and of extracellular ATP on ciliated rabbit trachea. The results are discussed with regard to the ciliary and calcium stimulation.     

Analysis of three-dimensional ciliary beating by means of high-speed stereomicroscopy.

Results are presented on the analysis of three-dimensional motion of compound cilia or cirri in voltage-clamped specimens of the protozoan Stylonychia mytilus. Time series of three-dimensional data were obtained by using the anaxial illumination method for simultaneous recording of stereoscopic video images. Data processing involved the following steps: determination of a reference coordinate system based solely on features present in each stereo-pair; tracing of cirral axes in digitized images, conversion to parameter curves by means of least-squares polynomial approximation, conversion of pairs of two-dimensional data to a series of three-dimensional data; correction for distortion due to projective shortening and conversion to a series of polynomial triplets, and analysis of the periodical components of the motion pattern in the frequency domain. Reconstructed beating cycles show typical differences between hyperpolarization-induced ciliary activity and depolarization-induced ciliary activity. Reconstructions of the motion of the basal segment of a cirrus are in agreement with existing data. Analysis of the curvature and torsion of a cirral axis during beating does not reveal any simple pattern of propagated activity within the axoneme. The return stroke may be subdivided into two phases. First, a curvature peak develops proximally. Secondly, a region with increased torsion arises more distally and spreads out in proximal direction. Both curvature and torsion return to minimal values by the beginning of the power stroke.     

Spectral characterization of ciliary beating. Temperature dependence on spectral parameters

Ciliary beating was optically examined in tissue cultures from frog palate epithelium. Consecutive segments of the analog signal were Fast-Fourier transformed. The three main parameters which define the spectrum, position of the peak maximum (f), width of the spectral line (S.D.), and area (A) under the spectrum, were all measured as a function of temperature. These measurements were also examined as a function of the number of cilia by varying the examined area from 1.2 to 122 microns2. It was found that: all the parameters were exponentially temperature dependent; and the average frequency was independent of the number of cilia examined, while S.D. was dependent on it. On a physiological level, we demonstrated that the ciliary fluctuation in frequency is temperature dependent, increasing with increase in temperature. Moreover, it was shown that where a relatively small number of cilia were measured (d = 1.24 micron), the area A under the observed spectrum was directly proportional to the amplitude of ciliary beating. Increasing the temperature decreases the amplitude and vice versa. According to our suggested model the dependence of A on f was predicted and verified experimentally. A mathematical model which simulates the S.D. as a function of examined area and temperature is suggested. The calculated results from the model are in a good agreement with our experimental findings.     

Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating.

Pseudomonas aeruginosa rhamnolipid causes ciliostasis and cell membrane damage to rabbit tissue, is a secretagogue in cats, and inhibits epithelial ion transport in sheep tissue. It could therefore perturb mucociliary clearance. We have investigated the effect of rhamnolipid on mucociliary transport in the anesthetized guinea pig and guinea pig and human respiratory epithelium in vitro. Application of rhamnolipid to the guinea pig tracheal mucosa reduced tracheal mucus velocity (TMV) in vivo in a dose-dependent manner: a 10-microgram bolus caused cessation of TMV without recovery; a 5-micrograms bolus reduced TMV over a period of 2 h by 22.6% (P = 0.037); a 2.5-microgram bolus caused no overall changes in TMV. The ultrastructure of guinea pig tracheal epithelium exposed to 10 micrograms of rhamnolipid in vivo was normal. Application of 1,000 micrograms/ml rhamnolipid had no effect on the ciliary beat frequency (CBF) of guinea pig tracheal rings in vitro after 30 min, but 250 micrograms/ml stopped ciliary beating after 3 h. Treatment with 100 micrograms/ml rhamnolipid caused immediate slowing of the CBF (P less than 0.01) of human nasal brushings (n = 7), which was maintained for 4 h. Mono- and dirhamnolipid had equivalent effects. The CBF of human nasal turbinate organ culture was also slowed by 100 micrograms/ml rhamnolipid, but only after 4 h (CBF test, 9.87 +/- 0.41 Hz; control, 11.48 +/- 0.27 Hz; P less than 0.05, n = 6), and there was subsequent recovery by 14 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bursting, beating, and chaos in an excitable membrane model.

We have studied periodic as well as aperiodic behavior in the self-sustained oscillations exhibited by the Hodgkin-Huxley type model of Chay, T. R., and J. Keizer (Biophys. J., 1983, 42:181-190) for the pancreatic beta-cell. Numerical solutions reveal a variety of patterns as the glucose-dependent parameter kCa is varied. These include regimes of periodic beating (continuous spiking) and bursting modes and, in the transition between these modes, aperiodic responses. Such aperiodic behavior for a nonrandom system has been called deterministic chaos and is characterized by distinguishing features found in previous studies of chaos in nonbiophysical systems and here identified for an (endogenously active) excitable membrane model. To parallel the successful analysis of chaos in other physical/chemical contexts we introduce a simplified, but quantitative, one-variable, discrete-time representation of the dynamics. It describes the evolution of intracellular calcium (which activates a potassium conductance) from one spike upstroke to the next and exhibits the various modes of behavior.     

Flagellar and ciliary beating in trypanosome motility

The single flagellum of Leishmania and Trypanosoma parasites is becoming an increasingly attractive model for the analysis of flagellar function-driven largely by the abundance of genomic and proteomic information available for the organelle, the genetic manipulability of the organisms and the importance of motility for the parasite lifecycle. However, as yet, there is a paucity of published data on the beating of any genetically malleable trypanosomatid species. Here we undertook an in-depth analysis using high-speed videomicroscopy of the beating of free-swimming Leishmania major cells in comparison to Crithidia species (for which there is some existing literature). In so doing, we describe a simple and generally-applicable technique to facilitate the quantitative analysis of free-swimming cells. Our analysis thoroughly defines the parameters of the expected tip-to-base symmetrical flagellar beat in these species. It also describes beat initiation from points other than the flagellum tip and a completely different, base-to-tip highly-asymmetric beat that represents a ciliary beat of trypanosomatid flagella. Moreover, detailed analysis of parameter interrelationships revealed an unexpected dependency of wavelength on oscillator length that may be the result of reversible constraint of doublet sliding at the tip or resonance of the flagellar beat.     

Hyperactivation is the mode conversion from constant-curvature beating to constant-frequency beating under a constant rate of microtubule sliding

Flagellar beating of hyperactivated golden hamster spermatozoa was analyzed in detail using digital image analysis and was compared to that of nonhyperactivated (activated) spermatozoa in order to understand the change in flagellar beating during hyperactivation and the active microtubule sliding that brought about the change in flagellar beating. Hyperactivated flagellar beating, which was characterized by a sharp bend in the proximal midpiece and low beat frequency, was able to alter the waveform with little change in beat frequency (constant-frequency beating), whereas activated flagellar beating, which was characterized by a slight bend in the proximal midpiece and high beat frequency, was able to alter beat frequency with little change in the waveform (constant-curvature beating). These results demonstrate that flagellar beating of hyperactivated and activated spermatozoa were essentially different modes and that hyperactivation was the mode conversion from constant-curvature beating to constant-frequency beating. Detailed analysis of flagellar bends revealed that the increase in curvature in the proximal midpiece during hyperactivation was due to the increase in total length of microtubule sliding in a nearly straight region between bends, while the rate of microtubule sliding remained almost constant.     

Prostaglandins have limited actions on abnormalities of beating induced in cultured heart cells.

Prostaglandins are antiarrhythmic in a variety of situations including ischaemic arrhythmias, but the mechanisms involved are not known. In view of this, the protective actions of prostaglandins A2, E2, F1 alpha, F2 beta, and I2 against abnormalities of beating induced in cultured heart cells were investigated. Abnormalities of beating were induced in single cells by variety of agents including ouabain Ca++, K+, dinitrophenol (DNP), and toxic material from the jellyfish Cyanea. Abnormalities were assessed in terms of rate, rate range, subjective arrhythmic behaviour and percent cells beating. The prostaglandins (at 10(-7)-10(-5) M) were added with the arrhythmogenic agent to test for their ability to modify agent-induced beating abnormalities and were compared with lidocaine and quinidine. Prostaglandins alone had minimal direct effects on the cells and only minimally reduced responses to arrhythmogenic agents. The most protective prostaglandins, PGE2 and PGF1 alpha, tended to normalise beating behaviour most noticeably in DNP-treated cells, unlike lidocaine and quinidine which were effective against Ca++-induced changes while worsening those of K+. Thus, a general ability to protect disturbed cardiac cells is not seen with high concentrations of prostaglandins.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Calcium ion regulation of chirality of beating flagellum of reactivated sea urchin spermatozoa.

Near an interface, sea urchin spermatozoa swim almost in circles. The direction is usually clockwise at the lower surface of a coverslip and counterclockwise at the upper surface of a glass slide, when viewed from above. Examination of demembranated spermatozoa has shown that Ca2+ regulates the direction of the circular motion of spermatozoa reactivated with adenosine triphosphate (ATP). This finding suggests that Ca2+ changes the chirality of the three-dimensional bending waves of sperm flagella.