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Biomedical subjects

D C Spray

Publications and source records attributed to D C Spray.

At least 145 records · Page 8Linked to original sources
PubMed1989

Neuronal analysis of pharyngeal peristalsis in the gastropod Navanax in terms of identified motoneurons innervating identified muscle bands. I. Muscle band identifiability.

The neuronal basis of pharyngeal ingestion and swallowing can be conveniently studied in the marine mollusc Navanax inermis because of the small number of neurons involved in the behavior, reproducible identifiability of many individual neurons and simple muscle arrangement. The Navanax pharynx contains 3 approximately orthogonal muscle groups, circumferential, longitudinal and radial, arranged in well defined layers. Pharyngeal peristalsis involves sequential circumferential constriction, apparently with coordinated local pharyngeal expansion produced by radial muscle. Circumferential and longitudinal muscles consist of discrete, well defined bands. The number, position and arrangement of circumferential and longitudinal bands show little interanimal variability; these bands are individually identified by sequential number. Regional morphologic specializations of circumferential bands presumably facilitate peristalsis. Circumferential and longitudinal bands form a two dimensional reference system defining position on the pharyngeal surface. In the accompanying paper circumferential motor fields are described in terms of identified motoneurons innervating identified bands, and radial motor fields are located with respect to the coordinate system of overlying longitudinal and circumferential bands.

Animals↗
PubMed1989

Neuronal analysis of pharyngeal peristalsis in the gastropod Navanax in terms of identified motoneurons innervating identified muscle bands. II. Radial and circumferential motor fields.

The neuronal basis of pharyngeal ingestion and peristalsis was studied in the gastropod Navanax inermis. Radially and circumferentially oriented muscles produce expansion and constriction of the pharynx. Motor fields of 11 identified radial motoneurons and 13 identified circumferential motoneurons were determined with respect to circumferential and longitudinal muscle band coordinates by muscle movements, electromyography, antidromic stimulation and axonal anatomy. Activation of these identified motoneurons can account for all the elemental pharyngeal movements observed during feeding. Four motoneurons, each innervating most of radial muscle, can mediate ingestion. Three radial motoneurons with anterior motor fields can mediate anterior expansion during sealing of the pharyngeal lips around prey and during regurgitation. Ten circumferential motoneurons have small arciform motor fields, the distributions of which correspond to the regional specializations in circumferential band organization. Arciform constriction can center eccentric ingested prey within the pharyngeal lumen during peristalsis. Arciform constrictions could combine to form an annular constriction in peristalsis. Small, non-overlapping, circumferential motor fields maximize the number of independent annular units available to produce a fine peristaltic wave. Sphincters have more circumferential motoneurons with smaller motor fields; this innervation permits finer motor control. Radial motoneurons with posterior motor fields can produce expansion caudal to a circumferential constriction during peristalsis. Motor fields of regional radial motoneurons show greater interanimal variability than circumferential motor fields, which is correlated with a less essential role of radial motoneurons in peristalsis. Two circumferential motoneurons with giant posterior pharyngeal motor fields can mediate pharyngeal emptying either in swallowing or in regurgitation.

Action Potentials↗
PubMed1989

Ionic coupling and mitotic synchrony of siblings in a Drosophila cell line.

Following mitosis in many cell lines, siblings remain adjoined in dyads until further cell division. We report here a series of experiments designed to ascertain the nature of this apposition in the embryonic Kc cell line of Drosophila melanogaster. We have found that (1) cell division in siblings is highly synchronized when compared to that in nonsiblings: (2) siblings in dyads are dye coupled with respect to Lucifer Yellow, but intercellular diffusion of larger molecules (FITC-dextran at 6 and 24 kDa) is retarded: (3) siblings are electrically coupled by an ungated low-resistance intercellular connection which is resistant to treatment with octanol and CO2, both known to close gap junction channels: and (4) members of a dyad are joined by a cytoplasmic bridge. Structures resembling septate junctions are also found between siblings and between cells in aggregates. The evidence accumulated here suggests that cytokinesis in Kc dyads is incomplete, resulting in an intercellular pathway that may provide for the passage of a molecular or electrical signal that regulates subsequent mitosis.

Animals↗
PubMed1989

Differential expression of three gap junction proteins in developing and mature brain tissues.

By using antibodies directed against gap junction proteins of liver (connexins 26 and 32) and heart (connexin 43), we have localized immunoreactivity to specific cell types in frozen sections of adult rodent brains. Connexin 32 reactivity was found in oligodendrocytes and also in a few neurons, whereas reactivity to connexins 26 and 43 was localized to leptomeningeal cells, ependymal cells, and pineal gland. Immunoreactivity with antibodies to connexin 43 also occurred in astrocytes. Furthermore, during embryonic and postnatal maturation of brain tissues, gap junction proteins were differentially expressed. Connexins 43 and 26 predominated in the neuroepithelium of embryonic brains, whereas connexin 32 was virtually absent. Between 3 and 6 weeks after birth, connexin 26 largely disappeared from immature brain; this time course corresponded to the increased expression of connexin 32. Expression of connexin 43 remained high throughout embryonic and postnatal development. These findings demonstrate that gap junction expression in the brain is diverse, with specific cell types expressing different connexins; this cell-specific distribution may imply differences in the function of these intercellular channels in different loci and developmental stages.

Aging↗
PubMed1989

mRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study.

The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.

Animals↗
PubMed1989

Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5-trisphosphate, and to calcium ions.

Hepatocytes are well coupled by gap junctions, which allow the diffusion of small molecules between cells. Although gap junctions in many tissues are permeable to molecules larger than cAMP and in several preparations gap junctions pass cAMP itself, little direct evidence supports permeation by other second-messenger species. Ca2+, perhaps the smallest second messenger, would be expected to cross gap junctions, but the issue is complicated because gap-junction channels are closed when intracellular free Ca2+ concentration, [Ca2+]i, is elevated to micromolar levels or above. Inositol 1,4,5-trisphosphate (InsP3), a second messenger that can evoke Ca2+ release, might also reduce junctional permeability by this mechanism. We report here evidence for transjunctional flux of Ca2+ and InsP3 in freshly isolated pairs or small clusters of rat hepatocytes. The Ca2+ indicator fura-2 was used to monitor transjunctional diffusion of Ca2+ directly or to detect passage of InsP3 by localized Ca2+ release. Fura-2 injected as the free acid passed between cells. Injection of InsP3 or CaCl2 immediately increased [Ca2+]i in the injected cell (peak values less than 1 microM), and [Ca2+]i increased rapidly in contacting cells (within seconds). The initial rise in [Ca2+]i induced by InsP3 was greater at discrete regions in the cytoplasm of both injected and uninjected cells and was inconsistent with simple diffusion of Ca2+. In the coupled cells the regions of greatest increase were not necessarily near the contact zone. In contrast, the rise induced in [Ca2+]i by CaCl2 injection when cells were bathed in normal Ca2+ was always more diffuse than with InsP3 injection, and in cells coupled to a cell injected with CaCl2 the earliest and maximal increases occurred at the region of cell contact. This difference in distribution indicates that injected InsP3 (or an active metabolite, but not Ca2+) diffused between cells to cause localized release of Ca2+ from intracellular stores. Ca2+ injection induced a rise in [Ca2+]i in coupled cells even when cells were maintained in Ca2+-free saline, suggesting that changes in [Ca2+]i seen in adjacent cells were due to transjunctional diffusion from the injected cell and not to uptake from the extracellular solution. However, in Ca2+-free saline, [Ca2+]i distribution was nonuniform, indicating that Ca2+-releasing mechanisms contribute to the observed changes. No increase in [Ca2+]i was seen in adjacent cells when Ca2+ was injected after treatment with the uncoupling agent octanol (500 microM), which itself did not change [Ca2+]i. These data provide evidence that the second messengers Ca2+ and InsP3 can be transmitted from cell to cell through gap junctions, a process that may have an important role in tissue function.

Animals↗
PubMed1989

Junctional communication is induced in migrating capillary endothelial cells.

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.

Adrenal Cortex↗
PubMed1989

cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Volatile anesthetics block intercellular communication between neonatal rat myocardial cells.

The effects of halothane and ethrane on gap junction-mediated intercellular communication and on membrane excitability were examined in cultured neonatal rat cardiac myocytes using whole-cell voltage-clamp and current-clamp techniques. Excitability was maintained at doses of both anesthetics that reversibly abolished current flow through junctional membranes. The degree of reduction of junctional conductance was a steep function of the dose of anesthetic; complete block occurred at lower aqueous concentrations of halothane than ethrane. The time course for loss of communication was rapid; 90% reduction of initial junctional conductance occurred in less than 15 seconds after exposure to 2 mM halothane or 4 mM ethrane. Recovery of junctional conductance and junctional permeability to intracellularly injected Lucifer yellow was rapid and complete on washout of the anesthetics. As junctional conductance was reduced by halothane or ethrane exposure, unitary conductance of the gap junctional channels remained constant at about 50 pS. Uncoupling by these anesthetics is thus attributable to a decrease in the number of conducting channels rather than to reduction of the channel's unitary conductance. The data are discussed with regard to the possible role of this intercellular communication pathway in the arrhythmias and alterations of conduction velocity and contractility produced by volatile anesthetics.

Animals↗
PubMed1988

Stationary and non-stationary occurrences of miniature end plate potentials are well described as stationary and non-stationary Poisson processes in the mollusc Navanax inermis.

Protractor muscles in the gastropod mollusc Navanax inermis exhibit typical spontaneous miniature end plate potentials with mean amplitude 1.71 +/- 1.19 (standard deviation) mV. The evoked end plate potential is quantized, with a quantum equal to the miniature end plate potential amplitude. When their rate is stationary, occurrence of miniature end plate potentials is a random, Poisson process. When non-stationary, spontaneous miniature end plate potential occurrence is a non-stationary Poisson process, a Poisson process with the mean frequency changing with time. This extends the random Poisson model for miniature end plate potentials to the frequently observed non-stationary occurrence. Reported deviations from a Poisson process can sometimes be accounted for by the non-stationary Poisson process and more complex models, such as clustered release, are not always needed.

Action Potentials↗
PubMed1988

Single-channel events and gating behavior of the cardiac gap junction channel.

The activity of gap junction channels between pairs of neonatal rat heart cells in culture was studied under control conditions and during uncoupling procedures by using dual whole-cell voltage clamp techniques. Under control conditions gap junctional conductance ranged from 0.05 to 35 nS. In cell pairs exhibiting low gap junctional conductance (less than 500 pS), single-channel events with a unitary conductance of 53 +/- 2 pS (5 experiments; 186 events) were apparent. Event duration and open-time probability were estimated to be 0.95 sec and 0.17, respectively. When the junctional conductance in well-coupled cell pairs (with initial junctional conductance, greater than 5 nS) was reduced by cytoplasmic acidification or application of heptanol, single-channel events could be visualized. Compared to low-conductance controls, unitary channel conductance was unaltered (for acidification the conductance was 58 +/- 3 pS in 11 experiments with 253 events; for heptanol the conductance was 61 +/- 1 pS in 2 experiments with 171 events), while the probability of channels being open was decreased. The constancy of unitary channel conductance under control conditions and during uncoupling procedures suggests that opening and closing of the gap junction channel are all-or-none processes during which no stable subconductance states are formed.

Alcohols↗
PubMed1988

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid.

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

Animals↗
PubMed1988

Cell uncoupling and protein kinase C: correlation in a cell line but not in a differentiated tissue.

Second messengers have been implicated in the control of communication between cells of various tissues and of a number of cell lines. To assess whether protein kinase C (PKC) is involved in the regulation of gap junctions between primary differentiated cells, we studied the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on PKC translocation and junctional conductance of rat pancreatic exocrine cells. Our results show that although TPA induced the translocation of PKC from a "cytosolic" to a "microsomal" fraction within minutes, it failed to block the junctional conductance of acinar cell pairs up to 30 min after application. By contrast, analogous experiments on a liver-derived cell line (WB cells) showed that TPA-induced PKC translocation was paralleled by a marked and irreversible inhibition of intercellular coupling. These results indicate that, in contrast to the effects on transformed or dedifferentiated permanent cell lines, PKC is not involved in gating gap junctional channels between primary differentiated secretory cells of the pancreas.

Animals↗
PubMed1988

Inotropic agents modulate gap junctional conductance between cardiac myocytes.

Cardiac gap junction channels mediate the intercellular exchange of second messenger and small molecules. Through effects on conduction velocity, enhanced junctional conductance (gj) facilitates rapid and synchronous activation of the contractile myocardium, whereas reduced gj slows activation and could contribute to arrhythmogenesis. We report here that gj is enhanced by agents that elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP; 8-bromo-cAMP or isoproterenol) and is depressed by agents that elevate intracellular guanosine 3',5'-cyclic monophosphate (cGMP; 8-bromo-cGMP or carbachol). Both effects occur with a time course comparable to the inotropic events mediated by these agents. The effect of cAMP on gj is not dependent on simultaneous changes in intracellular calcium; however, during calcium-overload conditions cAMP can precipitate calcium-dependent uncoupling. These results indicate that cyclic nucleotide-dependent changes in gj may contribute to the inotropic effects of these agents. Furthermore, the results suggest that the inotropic effect of cAMP includes a calcium-independent component.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Electrotonic synapses between Aplysia neurons in situ and in culture: aspects of regulation and measurements of permeability.

Properties of electrotonic synapses between L14 neurons in the abdominal ganglion of the marine mollusc Aplysia californica were examined in situ and between unidentified buccal neurons maintained in tissue culture. In culture, depolarizing postsynaptic potentials in response to a train of action potentials showed apparent facilitation with increasing spike number, which was attributable to the low-pass filter properties of electrotonic transmission via gap junctions and to network properties. Gap junctional conductance (gj), calculated from current-clamp data or measured directly under voltage clamp, indicated no significant dependence of gj on transjunctional or inside-outside potential in situ or in culture. Octanol, a local anesthetic agent that reduces gj in many other systems, had no effect on gj between Aplysia neurons. The effect of intracellular acidification, a treatment that rapidly and reversibly uncouples a variety of cell types, reduced gj between Aplysia neurons but did not completely abolish it. The relationship between intracellular pH (pHi), measured with ion-sensitive microelectrodes, and gj was steeper in cultured neurons than in situ and was maximally reduced by 70-80%, as compared to 50% or less in situ at the lowest pHi values tested. The coupling coefficient (k) was reduced less by low pHi than was gj, which could be explained by a simultaneous increase in nonjunctional membrane resistance. Permeability properties of Aplysia electrotonic synapses to a variety of tracer molecules were also examined between identified L 14 neurons in situ and in dissociated buccal, abdominal, and bag neurons in culture. The fluorescent dyes Lucifer yellow, 6-carboxyfluorescein, and dichlorofluorescein (1.2-1.4 nm maximal diameters) did not spread detectably from an injected neuron to its electrically coupled neighbors, regardless of the strength of electrotonic coupling. However, the smaller tetraalkylammonium ions TMA and TEA (diameters 0.66 and 0.8 nm, concentrations measured with ion-selective electrodes), could be detected in neighboring cells within minutes. In culture, transfer of the tetraalkylammonium ions was slow and not easily detectable in cell pairs where gj was low (less than 20 nS). The permeability was as high as 10(-10) cm3/sec in situ and 10(-12) cm3/sec in culture, and values were roughly correlated with simultaneously measured values of gj. Electrotonic synapses in the nervous system of Aplysia, therefore, have a quantitatively different spectrum of sensitivities than has been found for gap junctions of other systems and appear to possess reduced permeability to tracer molecules.

Animals↗
PubMed1987

Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes.

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent beta-mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

Animals↗
PubMed1987

Variation of gap junction sensitivity to H ions with time of day.

Conductance of gap junctions between segments of the lateral axons of the crayfish nerve cord is reduced by cytoplasmic acidification. Simultaneous measurements of axoplasmic pH and junctional conductance at 09.00 and 12.00 h revealed a shift of about 0.25 pH units in the apparent pK of the crayfish electrotonic synapse. These findings indicate that gating mechanisms of gap junctions are not fixed properties, but may rather be modulated by other, possibly humoral, factors.

Animals↗