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Disease↗
Biomedical subjects
B F King
Publications and source records attributed to B F King.
Formation and differentiation of extraembryonic mesoderm in the rhesus monkey.
Differentiation of extraembryonic mesoderm in the rhesus monkey was studied from the epithelial penetration stage of implantation (stage 4) through the first week of postimplantation development (to stage 6). It was found that the first cells that appeared between the primitive endoderm (hypoblast) and trophoblast were separated from the latter by a basal lamina but appeared to be either loosely attached to the endoderm or to have been detached from it. Cells in this intermediate position differentiated cytologically into mesenchymal cells, which, by stage 5, had a distinctive intraendoplasmic reticulum marker. This differentiation occurred prior to the time at which the primitive streak could be recognized. By the time the primitive streak was readily discernible (stage 6), the extraembryonic mesoderm had already produced substantial extracellular matrix. The sequence of differentiation was repeated, with a 1- to 2-day lag, in the secondary implantation site. No evidence of a contribution from cytotrophoblast or primitive streak to the extraembryonic mesoderm was found. It is concluded that the origin of the first extraembryonic mesoderm in the rhesus monkey is probably a two-step process, with formation of a reticulum from primitive endoderm followed by differentiation in situ into mesenchymal cells. The first blood vessels formed also differentiated in situ from the extraembryonic mesenchymal cells. Primitive capillaries were identifiable as early as the 13th day of pregnancy.
Animals↗
Central origin of vagal nerve fibres innervating the fundus and corpus of the stomach in rat.
The origin of vagal nerve fibres innervating the anterior and posterior walls of the fundus and corpus of the rat stomach was investigated using the axon tracing dye, Fast blue. The secretomotor nerve supply to the rat stomach was predominantly ipsilateral. A large majority (98-99%) of the vagal perikarya innervating the anterior fundus and corpus were located on the left side of the brainstem. A large majority (96-99%) of the vagal perikarya innervating the posterior fundus and corpus were located on the right side. Vagal perikarya were arranged in longitudinal, dorsal cell columns which extended beyond the normally accepted cytoarchitectural limits of the dorsal motor nucleus of the vagus (DMV). A few vagal cells innervating the fundus were also found in the nucleus ambiguus. Vagal cell columns innervating the anterior and posterior fundus extended rostrocaudally over a distance of up to 4 mm and projected caudally as far as the cervical spinal cord. Vagal cell columns innervating the anterior and posterior corpus were more compact, extended over a distance of 2-3 mm, and projected rostrally as far as the inferior salivatory nucleus of the glossopharyngeal nerves. Vagal cell columns for the fundus and corpus overlapped in the region of the DMV which lay immediately ventral to the area postrema. Between one-third to one-half of the vagal cells innervating the fundus and corpus were concentrated under the area postrema. A simple form of viscerotopic organisation appears to occur within the vagal cell columns innervating the fundus and corpus.
Animals↗
Immunohistochemical localisation and electrophysiological actions of GABA in prevertebral ganglia in guinea-pig.
Immunohistochemical techniques were used to detect the presence of a GABA-like material in prevertebral sympathetic ganglia in guinea-pigs. Varicose, immunolabelled nerve fibres were observed in close proximity to sympathetic neurones in inferior mesenteric ganglia and coeliac ganglia. Non-varicose, immunolabelled nerve fibres were observed in lumbar colonic nerves and superior coeliac nerves, i.e. in nerve bundles peripheral to prevertebral ganglia. Immunolabelling was also present in neurones in the myenteric plexus and in nerve fibres in the circular muscle of the colon, as shown previously (Hills et al., Neuroscience, 22 (1987) 301-312). However, GABA-like immunoreactivity was not observed in the cell bodies of prevertebral ganglia nor in splanchnic nerves central to prevertebral ganglia. It was concluded from these results that prevertebral ganglia in guinea-pig receive a GABAergic innervation from neurones peripheral to the ganglia, possibly from GABA-containing neurones in the myenteric plexus of the gastrointestinal tract. Intracellular recordings were made from sympathetic neurones in the inferior mesenteric ganglion (IMG). Application of GABA onto the IMG caused a slow depolarisation of sympathetic neurones, during which there was a marked decrease in the input resistance of IMG cells. Application of GABA also depressed excitatory postsynaptic potentials (EPSPs) and action potentials in sympathetic neurones excited by cholinergic nerve fibres in the lumbar colonic nerves. The reversal potential of the GABA-induced slow depolarisation was -37 mV, a value close to the chloride equilibrium potential for sympathetic neurones. The actions of GABA were reversibly reduced by the GABAA antagonist, bicuculline, and were modulated in a predictable manner by substituting chloride ions with methane-sulphonate ions. These results indicated that GABA, and presumably GABAergic nerves in prevertebral ganglia, modulate the excitability of sympathetic neurones by acting on GABAA receptors linked to a chloride ionophore.
Animals↗
Electronic characteristics and membrane properties of neurons in the inferior mesenteric ganglion in guinea-pig.
Intracellular recordings were made from neurons (n = 75) in the inferior mesenteric ganglion (IMG) in guinea-pig to study the electronic characteristics and membrane properties of IMG cells which receive an excitatory, cholinergic input from mechanoreceptors in the gastrointestinal tract. An excitatory, cholinergic innervation from the periphery served as an index to identify the sympathetic neurons involved in the reflex inhibition of muscle tone when the gut is distended. Functionally identified neurons in the IMG were categorized into 4 subclasses (I, II, III and IV). Subclasses I and II comprised neurons which fired phasically (rapidly adapting), with the neurons in subclass II showing a voltage relaxation in the electronic potentials elicited by depolarizing current-clamps. Subclasses III and IV comprised neurons which fired tonically (slow adapting), with the neurons in subclass III also showing relaxation of electronic potentials. Active and passive membrane properties were determined for neurons in each of the 4 subclasses of IMG cells. Measured values for the charging time-constant, the threshold current and the voltage threshold for firing (as well as calculated values for the input capacitance, specific membrane resistance, total surface area, cell diameter and cell space-constant) distinguished the neurons classed as phasic-firing from the neurons classed as tonic-firing. There were no statistical differences between the membrane properties of subclass I and II phasic neurons, or the membrane properties of subclass III and IV tonic neurons, to explain why the neurons in subclasses II and III showed a relaxation in electrotonic potentials during current-clamp. In the light of recent voltage-clamp data on the IMG cells the actions of time conductances for potassium ions are discussed to account for the variations in the electrotonic behavior of these IMG cells.
Action Potentials↗
Effects of potassium channel-blocking agents on neurons in the inferior mesenteric ganglion in guinea-pig.
Intracellular recordings were made from neurons (n = 121) in the inferior mesenteric ganglion (IMG) in guinea-pig. The pharmacological actions of 4-aminopyridine (4-AP), barium ions (Ba2+) and tetraethylammonium ions (TEA) were studied on IMG cells which received an excitatory, cholinergic input from mechanosensory nerves in the gastrointestinal tract. 4-AP mediated an excitatory action which involved two separate effects. Firstly, 4-AP increased the incidence of spontaneously occurring, fast EPSPs which gave rise to a spontaneous discharge of action potentials. This indirect, excitatory effect was attributed to an increase in the spontaneous release of acetylcholine from excitatory nerves to the IMG. Secondly, 4-AP altered the excitability of IMG cells and brought about burst discharges and continuous discharges of action potentials. This direct, excitatory effect was not dependent on the spontaneous release of acetylcholine; instead, it was attributed to the blockade of a potassium current similar to the A-current (IA). The excitatory action of Ba2+ also involved two separate effects. Firstly, Ba2+ increased the incidence of spontaneously occurring, fast EPSPs which gave rise to a spontaneous discharge of action potentials. This indirect, excitatory effect was interpreted as Ba2+ mimicking the actions of Ca2+ to facilitate the spontaneous release of acetylcholine. Secondly, Ba2+ altered the excitability of IMG cells and brought about a continuous discharge of action potentials. This excitatory effect was attributed to the blockade of a potassium current similar to the M-current (IM). TEA exerted an excitatory, then inhibitory, action on IMG cells. Initially, TEA brought about the continuous discharge of action potentials; firing gradually arrested as IMG cells depolarized slowly and a depolarizing block of excitation (i.e. inhibition) developed. The block on excitation was relieved by first restoring the resting membrane potential of IMG cells with hyperpolarizing current-clamp. Thereafter, action potentials were elicited by anode-break excitation by temporarily removing the hyperpolarizing current-clamp. The durations of action potentials and afterspike hyperpolarizations were prolonged in the presence of TEA. The effect of TEA on the action potential of IMG cells was attributed to the blockade of the delayed rectifier (IK). The effect on the afterspike hyperpolarization was considered the indirect consequence of a blockade of IK; it allowed the development of an inward calcium current which enhanced the calcium-activated, potassium current (IKCa) mediating afterhyperpolarizations.
4-Aminopyridine↗
Afterspike-hyperpolarization of neurons in the inferior mesenteric ganglion in guinea-pig.
Intracellular recordings were made from neurons (n = 121) in the inferior mesenteric ganglion (IMG) in guinea-pig. The afterspike hyperpolarization (ASH) following a single action potential was studied in IMG cells which received an excitatory, cholinergic innervation from mechanosensory nerves in the gastrointestinal tract. The amplitude of ASH was dependent on the membrane potential of IMG cells and the concentration of K+ in the bathing solution. The reversal potential of ASH (-80- -90 mV, in normal Krebs solution) appeared to follow the equilibrium potential for K+, as [K+]o was changed, suggesting that ASH was the product of K+-efflux. Further evidence suggested that a major component of the K+-efflux was dependent on the concentration of Ca2+ in the bathing medium. Elevation and reduction of [Ca2+]o increased and decreased, respectively, the amplitude and duration of ASH. In the presence of tetrodotoxin, depolarizing current pulses elicited spike-like events which (1) were dependent on [Ca2+]o and the degree of depolarization by current-clamp and (2) were followed by afterhyperpolarizations that were also dependent on [Ca2+]o and degree of depolarization by current-clamp. In the combined presence of tetrodotoxin and tetraethylammonium, depolarizing current pulses elicited prolonged action potentials (up to 100 ms in duration) followed by prolonged ASH (up to 3 s in duration). Spike-like events, prolonged action potentials and their afterhyperpolarizations were reduced in amplitude and duration when the calcium-channel blocking ion, Co2+, or blocking drug, verapamil, was present in the bathing medium. In normal Krebs solution, the ASH of action potentials produced by nerve stimulation was reduced but not abolished in the presence of Co2+. These results suggested that Ca2+ entered IMG cells during depolarization and activated the K+-conductance mechanisms responsible for the ASH. However, an initial component of the ASH may have involved other voltage-dependent K+-currents known to be activated during the excitation of sympathetic neurons. The amplitude and duration of ASH differed during non-synaptic and synaptic excitation of IMG cells, and differed when action potentials resulted from fast and slow EPSPs. In addition, the amplitude and duration of ASH were altered by noradrenaline, by the cholinomimetic, carbachol, and by 3 neuropeptides present in the IMG, namely leucine-enkephalin, substance P and vasoactive intestinal polypeptide.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Distribution of physostigmine and metabolites in brain subcellular fractions of the rat.
The distribution of 3H-physostigmine (Phy) has been studied in the rat brain subcellular fractions at various time intervals following i.v. injection. 3H-Phy or its metabolites rapidly accumulate into the cytoplasm of cells and penetrates the intracellular compartments. Kinetic studies of the subcellular distribution of radioactivity (RA) per gm of rat brain following i.v. injection of 3H-Phy show peak concentrations at 30 min in all subcellular fractions with the exception of mitochondria. In the mitochondrial fraction the RA levels continue to rise from 4682 +/- 875 DPM/gm at 5 min to 27474 +/- 2825 DPM/gm at 60 min (P less than .05). The cytosol contains the highest RA: 223341 +/- 21044 DPM/gm at 30 min which declined to 53475 +/- 3756 DPM/gm at 60 min. RA in synaptosome, microsomes and myelin increases from 5 to 30 min, and declines at 60 min. In vitro studies did not show a greater uptake of RA by the mitochondrial or synaptosomal fractions. The finding of relatively high concentrations of RA in the mitochondrial fraction at 60 min increases the likelihood that Phy or its metabolites could interfere with the physiological function of this organelle.
Animals↗
Ultrastructural differentiation of stromal and vascular components in early macaque placental villi.
In this study, closely staged placental villi from rhesus monkeys between 19 and 60 days of gestation were used to examine 1) the origin of endothelial cells and the mechanism of angiogenesis in villi; 2) the origin of placental macrophages (Hofbauer cells), and 3) the origin of the reticular cells that compartmentalize the stroma. The results did not support the concept that early stromal cells in the villi were derived by in situ delamination from cytotrophoblast. The extraembryonic mesodermal (mesenchymal) cells at the earliest of ages examined contained considerable granular ER. These cells organized into groups and formed primitive intercellular junctions, thus giving rise to the early angioblastic masses. The angioblastic masses were cellular, not syncytial; and lumen formation was not the result of intracellular vacuolization, but rather was the result of the acquisition of junctionally defined spaces. The earliest capillaries lacked intravascular blood cells and a basal lamina. Later, blood cells were evident in the lumina. At about 45-50 days of gestation, fetal capillaries began to indent the basal surface of the trophoblast. The basal lamina of the fetal capillaries still had not developed by 60 days of gestation. Between 35 and 40 days of gestation, significant morphological changes took place in the villous stroma. Evidence was obtained that the mesenchymal cells differentiated into the reticular cells that subdivided the stroma into fibril-rich and fibril-free compartments. At the same time, Hofbauer cells were observed for the first time; they occupied the fibril-free regions of the stroma. We did not observe any clear indications of intermediate stages of differentiation between other stromal cell types and Hofbauer cells. It is suggested that placental macrophages may have an origin independent of other stromal types; one possibility is that they are derived from blood monocytes as in other tissues. It is further suggested that the activities of the macrophages and reticular cells may be important in remodeling the extracellular matrix and may be related to the process of branching morphogenesis in the villi.
Animals↗
Excitatory innervation of the rabbit rectococcygeus muscle by enteric nerves from the terminal large intestine.
The rectococcygeus muscle extends from the longitudinal layer of the external muscles of the terminal colon, anchoring the large intestine to the coccygeal vertebrae. The muscle is thought to assist the colon during defaecation, drawing the large intestine partly into the pelvic cavity. In the rabbit, many fine nerves run between the wall of the colon and a ganglionated nerve plexus on the ventral face of the rectococcygeus muscle. Extracellular recordings of evoked and spontaneously occurring multiunit discharges from these nerve bundles indicated that they may take their origin from the enteric nervous system of the large intestine. Enteric nerves (as we have called them) conducted bursts of spontaneously occurring multiunit discharges, each burst associated with a twisting movement of the rectococcygeus and the colon. Electrical stimulation of enteric nerves elicited cholinergically mediated contractions of the rectococcygeus; however, nerve stimulation failed to elicit relaxation of muscle tone induced by carbachol. Therefore, enteric nerves appeared to supply the rectococcygeus with an excitatory innervation only. These excitatory nerves appeared to remain uninterrupted by a ganglion synapse as they travelled to the rectococcygeus. Stimulation of enteric nerves caused the left and right hemirectococcygeal muscle strips to twist and deform the colo-rectal region to which they were attached. From these results, it is suggested that the rectococcygeus may delay the movement of luminal contents from colon to rectum by forming a weak valve which partially occludes the colo-rectal region. The enteric nerves to the rectococcygeus may assist in the maintenance of continence.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Sorting and transepithelial transport of adsorbed protein tracers: effects of temperature.
The epithelium of the guinea pig yolk sac is involved in the selective transport of macromolecules to the fetus. We studied the compartments involved in sorting and transepithelial transport of protein tracers and the effect of lowered temperature (18 degrees C) on these events. Explants of yolk sac were incubated with a mixture of cationized ferritin (CF) and horseradish peroxidase (HRP, Sigma type VI). At 4 degrees C, both tracers were bound to the cell surface and binding of an HRP-gold complex was shown to be inhibited by mannan. At 37 degrees C and 18 degrees C, both tracers were taken up into tubules and vesicles in the apical cytoplasm. Usually the tubules contained a mixture of tracers, but they often showed a polarized distribution with CF and HRP at opposite ends. The vesicles also contained mixtures of the tracers, but some contained only one. In addition, there were some irregularly shaped vacuoles composed of saccules that contained either a mixture, HRP alone, or CF alone. These results suggest that these adsorbed ligands are binding to unique microdomains of the endocytic complex. After 20 min at 37 degrees C coated vesicles 100 nm in diameter were located in the apical cytoplasm and coated vesicles of the same size were located at the lateral cell membrane. Usually they contained only HRP or CF, although occasional mixtures were seen. At 18 degrees C, HRP was transported across the cells in 100 nm vesicles. However, transport of CF was completely inhibited at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cardiac tamponade from chronic pericardial effusion in hypothyroidism.
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Aged↗
Endocytosis and subsequent processing of 125I-labelled immunoglobulin G by guinea pig yolk sac in vitro.
We have developed conditions for studying the binding, uptake, degradation and transport of 125I-labelled IgG by yolk sac in vitro. Specific binding to tissue at 4 degrees C and to paraformaldehyde-treated tissue at 37 degrees C was time- and temperature-dependent and showed saturation kinetics (Kd,4 degrees C = 2.9 X 10(-6) M, Kd,37 degrees C = 5.3 X 10(-6) M). Uptake was studied at 37 degrees C using untreated tissue (K uptake = 13.3 X 10(-6) M) and was inhibited by preincubation with metabolic poisons but not with cycloheximide. Tissue that had been incubated with 125I-labelled IgG at 37 degrees C released radiolabelled degradation products and intact 125I-labelled IgG into the medium. Experiments with paraformaldehyde-treated and untreated tissue showed that release of intact 125I-labelled IgG was mostly the result of ligand dissociation from surface binding sites. However, more 125I-labelled IgG was released from untreated tissue than could be accounted for solely by loss of surface-bound ligand and the difference was presumed to reflect uptake, transport and exocytosis of 125I-labelled IgG. Degradation of 125I-labelled IgG was inhibited by leupeptin and lysosomotropic amines. These drugs had no detectable effect on 125I-labelled IgG release. The results suggest that degradation and transport of IgG are not intimately related and are consistent with a previously proposed model for IgG transport via coated vesicles which do not fuse with lysosomes and for non-selective uptake into another class of vesicle which does fuse with lysosomes.
Adenosine Triphosphate↗
Transport of horseradish peroxidase across monkey trophoblastic epithelium in coated and uncoated vesicles.
This study used membranous chorion of the macaque monkey placenta to examine uptake and processing of exogenous proteins. Tissue was incubated with either cationic or anionic horseradish peroxidase. Incubation time was varied between 5-25 min to follow the endocytic pathways. In spite of some differences in binding, uptake and processing of the isozymes was similar. In the presence of tracers at 37 degrees C both horseradish peroxidases were taken up in large (150-175) nm diameter) coated vesicles. In addition, coated tubules 300-400 nm in length and 50-100 nm in diameter were seen in the apical cytoplasm. Studies using ruthenium red indicated that the coated tubules were derived from long coated invaginations of the free surface that pinch off into the apical cytoplasm. Often, the tubules bud off small (85-105 nm diameter) protein-filled coated vesicles which traversed the cytoplasm and fused with the basal-lateral plasma membrane. In other cases, the tubules or vesicles lost their clathrin coats and fused to form larger endocytic vesicles which later fused with phagolysosomes. After long incubation, larger uncoated vesicles (endosomes) were seen releasing their contents at the basal-lateral membrane. These results suggest that multiple transport pathways exist in this epithelium. The first, involving only coated structures, may function to sort and concentrate specific ligands important for embryonic development. The second, involving the formation and translocation of large uncoated vesicles to the basal-lateral membrane, may also provide nutrients to the embryo. A third pathway directs the protein to phagolysosomes where it is presumably degraded.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid Phosphatase↗
Ruthenium red staining of vaginal epithelial cells and adherent bacteria.
The vaginal epithelium of the rhesus monkey is a keratinized stratified squamous epithelium throughout the menstrual cycle and early pregnancy. The superficial cells have a thickened cell envelope, surface microridges, and numerous adherent bacteria. During later pregnancy the cells mucify and have a typical cell membrane, microvilli, and no adherent bacteria. In the present study we have extended these observations by examining vaginal surface structures after ruthenium red staining. Throughout the cycle, the superficial cells have a thin layer of stained material closely associated with the cell membrane, but in some cases a much thicker mucous blanket was observed. During later pregnancy the epithelial cells had a moderately thick, somewhat clumped ruthenium red-positive material associated with the cell membrane. Glycocalyx components of the surface of many bacteria also stained with ruthenium red. The adherence of many types of bacteria to the vaginal epithelial cells appears to be effected by the interaction of polyanionic components on the surface of both the bacterial and epithelial cells.
Animals↗
Distribution and characterization of anionic sites in the basal lamina of developing human amniotic epithelium.
We have studied the distribution of anionic sites in the basal lamina of developing human amniotic epithelium by using the cationic stain ruthenium red. Amnions at 7-12 weeks of gestation and at term contained ruthenium red-positive granules in a quasi-regular array on both the cellular and interstitial sides of the lamina densa. In order to characterize the anionic sites, small pieces of amnion were incubated in the presence or absence of either chondroitinase ABC, neuraminidase, Streptomyces hyaluronidase, or heparitinase in appropriate buffer systems. Incubation in the presence of heparitinase resulted in the complete disappearance of the basal lamina-associated granules, but other enzymes tested had no demonstrable effect on these granules. We conclude that the anionic sites associated with amnion basal lamina, and demonstrable with ruthenium red, consist of glycosaminoglycans rich in heparan sulfate, probably present as heparan sulfate proteoglycan. Because amniotic fluid has a low protein content and amniotic epithelium (at least at term) lacks tight junctions, we postulate that the heparan sulfate proteoglycan associated with the amnion basal lamina may have an important function as a permeability barrier to anionic macromolecules.
Amnion↗
Distribution and characterization of anionic sites in trophoblast and capillary basal laminas of human placental villi.
The distribution of anionic sites was studied in the trophoblastic and fetal capillary basal laminas of developing human placental villi with the cationic stain ruthenium red. At 7-12 weeks of gestation the trophoblastic basal lamina (TBL) contained ruthenium red-positive granules in a quasi-regular array throughout the lamina densa or sometimes concentrated at the interstitial surface of the lamina densa. The capillary basal lamina (CBL) (and anionic sites) were not present at this age. Anionic sites were also associated with collagen or reticular fibrils. At term, the TBL was largely devoid of anionic sites except for some distributed along its interstitial surface. The CBL was present in later gestation and sometimes had arrays of anionic sites. In order to characterize the anionic sites, minced pieces of villi were incubated in the presence or absence of either chondroitinase ABC, heparitinase, neuraminidase, or Streptomyces hyaluronidase in appropriate buffer systems. Incubation of early villi with heparitinase resulted in the disappearance of the TBL-associated sites. Chondroitinase ABC appeared to reduce staining of collagen-associated sites. In term villi, heparitinase removed those few sites still associated with the TBL but did not affect sites associated with the CBL or collagen. Chondroitinase ABC resulted in the disappearance of all anionic sites. In later gestation, a number of developmentally important macromolecules are transported across the trophoblast and enter the fetal capillaries. We conclude that the absence of an array of polyanionic sites from the term placenta TBL and the reduction in the amount of extracellular matrix intervening between the trophoblast and capillaries are adaptations to enhance the exchange of macromolecules across the placenta.
Anions↗
Ultrastructural localization of acid phosphatase in nonhuman primate vaginal epithelium.
The vagina of the rhesus monkey is lined by a stratified squamous epithelium. However, little is known regarding the cytochemical composition of its cell organelles and the substances found in the intercellular spaces. In this study we have examined the ultrastructural distribution of acid phosphatase in the vaginal epithelium. In basal and parabasal cells reaction product was found in some Golgi cisternae and vesicles and in a variety of cytoplasmic granules. Reaction product was also found in some, but not all, membrane-coating granules. In the upper layers of the epithelium, the membrane-coating granules extruded their contents and acid phosphatase was localized in the intercellular spaces. The possible roles of acid phosphatase in keratinization, desquamation, or modification of substances in the intercellular compartment are discussed.
Acid Phosphatase↗