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Mechanochemical coupling in flagella. IV. Absence of coupling between metabolism and motility in Chaetopterus spermatozoa.

The metabolic rate of suspensions of spermatozoa from the annelid worm, Chaetopterus, does not change significantly when the viscosity of the suspension is increased by the addition of methyl cellulose. At high viscosities, where the spermatozoa swim with about 1/3 the normal beat frequency and 1.5-1.8 times the normal bend angle, the calculated power output against external viscous resistances increases to 4-5 times its normal value. This large increase in power output can be accommodated without an increase in metabolic rate only if internal viscous resistances to flagellar bending are relatively low.

Animals↗

Coupling of iodotyrosine catalyzed by human thyroid peroxidase in vitro.

The coupling of iodotyrosine (coupling reaction) is one of the least studied in the formation of thyroid hormone, particularly in human thyroid diseases. This paper describes a method of measuring iodotyrosine coupling catalyzed by human thyroid peroxidase (TPO) in vitro. There were two important requirements to demonstrate the coupling reaction: 1) thyroglobulin with a low thyroid hormone content, and 2) partially purified TPO. Thyroglobulin with low thyroid hormone content was obtained from Grave's and follicular adenoma tissues after propylthiouracil (PTU) therapy and L-T4 therapy, respectively. TPO was prepared from Graves' thyroid by solubilizing the 100,000 X g pellet of thyroid homogenate with sodium deoxycholate and trypsin, followed by Sephacryl S-300 gel filtration. Before the coupling reaction, thyroglobulin was iodinated with chloramine-T and potassium iodide, followed by dialysis. The coupling reaction was carried out by incubating newly iodinated thyroglobulin with TPO, diiodotyrosine, a coupling stimulator, and a H2O2-generating system (glucose and glucose oxidase) for 20 min at 37 C. After thyroglobulin was digested with Pronase, the thyroid hormone content of the thyroid digest was measured by RIA. Coupling activity was measured by the amount of newly formed T3 (nanograms of T3 per mg thyroglobulin). The time course of coupling reaction showed a progressive increase in coupling activity up to 30 min, and the reaction was temperature and pH dependent, with a pH optimum of 7.0. Coupling activity in the presence of H2O2 and TPO was 43 +/- 5.0 ng T3/mg thyroglobulin (mean +/- SD of triplicate samples), and addition of diiodotyrosine to the H2O2-TPO system caused a nearly 3-fold increase in coupling activity. This method has potential utilization for measurement of peroxidase coupling activity, since there was a linear relationship between the measured coupling activity and the amount of added TPO when the TPO concentration was over 3 micrograms/300 microliter. Methimazole (MMI) and PTU had similar potencies in inhibiting the TPO-catalyzed coupling reaction, whereas MMI was distinctly more potent than PTU as an inhibitor of TPO-mediated iodination in vitro. The different potencies of MMI in the two reactions suggest that different inhibitory mechanisms may be involved in iodination and coupling. The reducing agent, sodium metabisulfite, was also found to be a more potent inhibitor of the TPO-mediated coupling reaction than of the TPO-mediated iodination reaction. The method of iodotyrosine coupling described here may be useful to investigate the coupling step of thyroid hormone formation in human thyroid diseases.

Catalysis↗

Localization of the Na(+)-coupled neutral amino acid transporter 2 in the cerebral cortex.

We studied the distribution and cellular localization of Na(+)-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na(+)-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na(+)-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na(+)-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na(+)-coupled neutral amino acid transporter 2. Na(+)-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na(+)-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na(+)-coupled neutral amino acid transporter 1- and Na(+)-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na(+)-coupled neutral amino acid transporter 1/synaptophysin and Na(+)-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na(+)-coupled neutral amino acid transporter 1 and Na(+)-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate-glutamine cycle. The strong expression of Na(+)-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood-brain and brain-cerebrospinal fluid barrier suggests that Na(+)-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.

Amino Acid Transport System A↗

Cell-cell coupling between CO2-excited neurons in the dorsal medulla oblongata.

Anatomically coupled neurons (17 of 137) and non-coupled neurons (120 of 137), in and near the nucleus tractus solitarius and dorsal motor nucleus (i.e. solitary complex), were studied by rapid perforated patch recording in slices (rat, 150-350 microm thick, postnatal day 0-21) before, during and after exposure to hypercapnic acidosis. Anatomical coupling refers to the intercellular transfer of Lucifer Yellow and Biocytin into adjoining neurons, presumably via gap junctions [see Dean et al. (1997) Neuroscience 80, 21-40]. Eighty-six per cent of the anatomically coupled neurons (12 of 14) were depolarized by hypercapnic acidosis, a response referred to as CO2 excitation or CO2 chemosensitivity. In all, 28% (12 of 43) of the CO2-excited neurons were anatomically coupled to at least one other neuron. None (0 of 39) of the CO2-inhibited neurons were anatomically coupled, and only 4% (two of 46) of the CO2-insensitive neurons were anatomically coupled. Increasing the fractional concentration of CO2 from five to 10 and 15% in constant bicarbonate (26 mM) decreased intracellular pH (control 7.3 7.4, 22-25 degrees C) by approximately 1.0 and 1.5 pH units, respectively, as measured using the pH-sensitive fluorescent dye, 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein. Nine of the anatomically coupled neurons (six CO2-excited, one CO2-insensitive and two unidentified) exhibited spontaneous electrotonic postsynaptic potential-like activity, suggesting that they were also electrotonically coupled. During hypercapnic acidosis, the amplitudes of electrotonic postsynaptic potentials were unchanged, concomitant with small changes in input resistance. The frequency of electrotonic postsynaptic potentials increased during hypercapnic acidosis in many anatomically coupled neurons (eight of nine), indicating that both neurons of the coupled pair were stimulated. Cell-cell coupling occurred preferentially in and between CO2-excited neurons of the solitary complex. Further, CO2-excited neurons were not electrotonically uncoupled during intracellular acidosis, in contrast to the effect that decreased intracellular pH has on many other types of coupled cells. It was not determined whether anatomical coupling was affected by hypercapnic acidosis since dye mixture was always administered under normocapnic conditions. The high correlation between anatomical coupling, electrotonic coupling activity and CO2-induced depolarization suggests that cell-cell coupling is an important electroanatomical feature in CO2-excited neurons of the solitary complex. CO2-excited neurons have been hypothesized to function in central chemoreception for the cardiorespiratory control systems, suggesting that cell cell coupling may contribute in part to central chemoreception of CO2 and H+.

Action Potentials↗

Functional coupling of G proteins to endothelin receptors is ligand and receptor subtype specific.

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins. 2. Coupling between endothelin A (ET(A)) or endothelin B (ET(B)) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein alpha-subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein alpha-subunits. 3. Unstimulated ET(A) and ET(B) receptors (ET(A)R and ET(B)R, respectively) were barely coupled to Go(alpha). The unstimulated ET(A)R coimmunoprecipitated with Gi3alpha, whereas the unstimulated ETBR was much less strongly coupled to Gi3alpha. The coupling of ETBR to Gi1Gi2 alpha-subunits was much stronger than the coupling of ET(A)R to these alpha-subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET(B)R to Gi3alpha, whereas coupling of the ET(A)R to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET(A)R to G(q)alpha/G11alpha than in the coupling of ET(B)R to these alpha-subunits. Ligand-induced coupling between the receptors and the Gi1 and Gi2 alpha-subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to Go(alpha), except that ET-3 increased the coupling of this alpha-subunit to ET(B)R and decreased the coupling to ET(A)R. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific. 4. It remains to be established whether this diversity of receptor-G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.

Animals↗

Direct evidence for electrical coupling among rat supraoptic nucleus neurons.

Transfer of the fluorescent dye, Lucifer yellow (LY), from an intracellularly injected neuron to one or more other neurons is accepted as indirect evidence of electrotonic interactions among such dye coupled cells. Direct evidence requires that at least two coupled cells be recorded from simultaneously and such evidence in the CNS has been gained only for hippocampal pyramidal neurons. Since interpretations of the functional significance of dye coupling among magnocellular neuroendocrine cells depend upon its relation to electrical coupling, we sought to obtain direct evidence for electrotonic interactions in such neurons. Over 150 pairs of supraoptic nucleus (SON) neurons in hypothalamic slices were recorded from intracellularly using one LY and one potassium acetate electrode in each instance. Of these, 9 pairs were studied in sufficient detail to determine that they were electrically coupled. Most of the remaining pairs were determined not to be coupled. In each coupled pair of cells, membrane voltage changes due to spontaneously occurring or current evoked action potentials, as well as current evoked hyperpolarizations, in one cell were reflected in similar, though attenuated changes in the other cell. All of these changes occurred simultaneously in the two neurons. Spontaneously arising postsynaptic potentials in the two cells were temporally uncorrelated. In each case that electrical coupling was observed, dye coupling resulted from LY injection. Coupling ratios ranged from 0.05 to 0.2. Capacitative coupling between the recording electrodes as an artifact was ruled out since cells in the same tissue penetration as the coupled cell showed no responses to membrane voltage changes in the primary cell; no responses were seen with the second electrode placed extracellularly or in the medium; and similar coupling potentials were also seen when one cell was recorded without a second electrode present. We conclude that electrical coupling exists among magnocellular neurons of the SON and that the incidence of dye coupling is a reasonable estimate of the incidence of electrical coupling. These electrotonic interactions probably play important roles in the coordination of firing among magnocellular neurosecretory neurons.

Action Potentials↗

Effects of prolonged hypobaric hypoxia on carotid nerve endings and glomus cells. Changes in intercellular coupling.

Carotid bodies were removed from anesthetized rats kept under normobaric (640 Torr) and hypobaric conditions (380 Torr for 2-3 weeks). Slices (100-150 microm) of the organ were viewed under an inverted microscope for simultaneous stimulation and recording of coupled glomus cells and carotid nerve endings. The latter were identified by their more negative Em, high input resistance (Ro) and time-dependent rectification in response to negative current pulses. Also, when nerve endings had an Em more negative than -40 mV showed spontaneous activity in the form of mini-receptor potentials (mrps). Glomus cells had less negative Em and lower Ro. Prolonged hypobaric hypoxia did not change the Em of nerve endings and glomus cells. However, in both structures, Ro increased. Also, the mrps became smaller and occurred less frequently. Intercellular coupling was recognized when currents applied to one cell spread to adjoining ones. In the case of glomus cells (GC/GC coupling), it was mostly resistive and bidirectional. Coupling between nerve endings and glomus cells was more complex, When a glomus cell was stimulated, current spread to the nerve ending (GC/NE coupling) was similar in magnitude (2-3%) to coupling between GCs. However, when NE was stimulated current spread to GC (NE/GC coupling) was minimal (less than 0.1%) and transient (capacitive). Nerve endings were also bidirectionally and capacitively coupled (NE/NE coupling) with a median of 2,8%. Intracellularly injected Lucifer Yellow or Alexa 488 diffused to neighboring structures. Prolonged hypobaric hypoxia significantly tightened coupling modes GC/NE, NE/GC, and NE/NE but reduced GC/GC coupling. Tighter coupling was accompanied by lower coupling resistance, and the opposite occurred when intercellular coupling decreased. Increased GC/NE and reduced GC/GC coupling during hypobaric hypoxia may be partly responsible for the increased reactivity of these receptors under this condition.

Animals↗

Intracellular calcium response and adhesiveness of chick embryonic retinal neurons to photoelectric dye-coupled polyethylene films as prototypes of retinal prostheses.

Photoelectric dyes, which absorb light and convert photon energy to electric potentials, were shown to stimulate retinal neurons in culture. One of these dyes coupled with polyethylene film surface, as a prototype of retinal prostheses, could induce intracellular calcium elevation in chick embryonic retinal tissues. In this study, we used retinal cells from chick embryos in which no photoreceptor outer segments yet developed, and assessed cell adhesiveness and response to the original and modified types of our retinal prostheses. Extruded-blown film of high density polyethylene was used as the original polyethylene film. Polyethylene film surface was modified either by coupling with type 1 collagen or by crystallization from the melt of the original polyethylene film. A photoelectric dye, 2-[2-[4-(dibutylamino)phenyl]ethenyl]-3-carboxymethylbenzothiazolium bromide, was coupled to these different types of polyethylene films through amide linkage. Retinal cells from chick embryos at the 12-day embryonic stage were cultured on the surface of dye-coupled films. The response of retinal cells to the dye-coupled films was observed by calcium imaging. Cell types, either neurons or glial cells, were detected by immunocytochemical staining and also observed by scanning electron microscopy (SEM). Intracellular calcium elevation was observed in cells, including both neurons and glial cells, on the surface of the original dye-coupled film, collagen-coated dye-coupled film, and dye-coupled recrystallized film, in contrast with no response of cells cultured on the dye-uncoupled plain film. Retinal neurons grew upon colonies of glial cells settling on the surface of all different types of the films. The number of retinal neurons on the collagen-coated dye-coupled film and dye-coupled recrystallized film was significantly larger than the number of neurons on the dye-uncoupled plain film (P = 0.0004, analysis of variance; P < 0.05, Tukey-Kramer test). With SEM, Muller cell-like large cells covered the original dye-coupled film and collagen-coated dye-coupled film, while neuron-like small cells settled directly on the dye-coupled recrystallized film and also formed large colonies on Muller-like cells. Retinal neurons and glial cells were cultured directly on the different types of dye-coupled polyethylene films. Photoelectric dye-coupled polyethylene films could be used as prototypes of retinal prostheses.

Aniline Compounds↗

Electrotonic coupling in the inferior olivary nucleus revealed by simultaneous double patch recordings.

Electrotonic coupling in the inferior olivary (IO) nucleus is assumed to play a crucial role in generating the subthreshold membrane potential oscillations in olivary neurons and in synchronizing climbing fiber input into the cerebellar cortex. We studied the strength and spatial distribution of the coupling by simultaneous double patch recordings from olivary neurons in the brain slice preparation. Electrotonic coupling was observed in 50% of the cell pairs. The coupling coefficient (CC), defined as the ratio between voltage responses of the post- and the prejunctional cell, varied between 0.002 and 0.17; most of the pairs were weakly coupled. In more than 75% of the pairs, the CC was <0.05. The coupling resistance varied between 0.7 to 19.8 G(Omega), and 68% of the values fell between 0.7 to 8 G(Omega). The difference between the coupling coefficient measured on stimulation of cell 1 or cell 2 of a coupled pair was 27 +/- 16%. Direct calculation of the coupling resistance revealed an asymmetry of 24 +/- 12%, suggesting a directional preference of coupling. The coupling was voltage independent, although depolarization of either the pre- or the postjunctional neuron reduced the CC. The chance of a cell pair being coupled was 80% in immediate neighboring cells, but dropped to about 30% at a distance of 40 microm. No coupled pairs were observed at distances larger than 70 microm. In 52% of staining experiments neurobiotin injection into an olivary neuron produced indirect labeling of 1-11 nearby cells with an average of 3.8 +/- 2.9. All indirectly labeled cells were found in, or immediately adjacent, to the dendritic field of the directly stained neuron. Two distinct morphological types of olivary neurons, "curly" and "straight" cells, were found. In each case all neurons stained indirectly by dye passage through gap junctions belonged to the same type. Using the physiological data we estimated that each olivary neuron is directly coupled to about 50 neurons. Since somatic recordings may not reveal coupling through remote dendrites, we conclude that each neuron is directly connected to > or =50 neurons forming two distinct networks of curly and straight cells.

Action Potentials↗