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  • 1
    Electronic Resource
    Electronic Resource
    Application of a new pH-sensitive fluoroprobe (carboxy-SNARF-1) for intracellular pH measurement in small, isolated cells (1990)
    Springer
    Pflügers Archiv 417 (1990), S. 234-239 
    Details
    ISSN: 1432-2013
    Keywords: Intracellular pH ; SNARF-1 ; Carotid body
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report the use of a new pH-sensitive dualemission fluoroprobe, carboxy-seminaphthorhodafluor-1 (carboxy-SNARF-1) for ratiometric recording of intracellular pH (pHi) in small isolated cells. The method is illustrated with pHi measurement in single type-1 cells (cell diameter ∼10 μm) isolated from the carotid body of the neonatal rat. Carboxy-SNARF-1 is loaded using bath application of the acetoxymethyl ester. When excited at 540 nm, the fluoroprobe gives strong, inversely related emission signals at 590 nm and 640 nm. Stable ratiometric recordings of pHi can be achieved from a single cell (pHi 8.5-6.5) for up to 50 min. Photobleaching of the probe is minimised by illuminating at relatively low light intensity (50 W xenon lamp with 0.2% transmission neutral density filter). The probe can be calibrated in situ using the nigericin technique and this is in good quantitative agreement with the independent null-point technique (extracellular weak acid/weak base application) of Eisner et al. (1989). This fluoroprobe offers certain advantages over the other commonly used probe for pHi 2′,7′-bis-(2-carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF): (i) because of its two strong pH-sensitive peak emissions, SNARF displays a good signal-to-noise ratio for ratiometric recording at low light intensities; (ii) unlike BCECF, the dual emisson of SNARF requires no sequential mechanical switching of excitation filters, thus simplifying the epifluorescence set-up; (iii) because carboxy-SNARF-1 emission signals are at the yellow/red end of the visible spectrum, fluorescent drugs like amiloride, ethyl-isopropyl-amibride (EIPA), 4,4′-diisothiocyanostilbene 2,2′-disulphonic acid (DIDS) and cinnamate analogues do not interfere with the pHi recording, even when used at high concentrations.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00370705
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  • 2
    Electronic Resource
    Electronic Resource
    Assessment of evidence for K+-H+ exchange in isolated type-1 cells of neonatal rat carotid body (1997)
    Springer
    Pflügers Archiv 434 (1997), S. 429-437 
    Details
    ISSN: 1432-2013
    Keywords: Key words pHi ; SNARF ; Carotid body ; Type-1 cell ; Potassium-hydrogen exchange ; K+-H+ exchanger (KHE) ; Nigericin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract  Intracellular pH (pHi) was measured in enzymically isolated, neonatal rat carotid body type-1 cells, using the fluorophore carboxy-SNARF-1 (AM-loaded), and using the nigericin technique for in situ fluorescence calibration (nigericin is a membrane-soluble K+-H+ exchanger). In CO2/HCO3 –-free media, inhibiting Na+-H+ exchange produced a prompt fall of pHi (background acid-loading), the rate of which was reduced by raising the extracellular K+ concentration, [K+]o. pHi recovery from an intracellular acid or alkali load was also sensitive to changes of [K+]o. These results are similar to those of Wilding et al. (J Gen Physiol 100:593–608, 1992), who proposed the existence of an acid-loading, K+-H+ exchanger (KHE) in the type-1 cell. However, when nigericin was not used for post-experimental calibration, and the superfusion system was flushed exhaustively with strong detergent, alcohol and distilled water, then background acid-loading was attenuated, and the K+ o sensitivity of pHi insignificant. Background loading was increased again, and K+ o sensitivity restored, when cells were monitored in a superfusion system which had previously been exposed to a single nigericin-calibration protocol (followed by a short system wash with strong detergent and distilled water). We conclude that the previously reported expression of KHE in carotid body type-1 cells is an artefact caused by nigericin contamination. We have therefore quantified the pHi dependence of background loading in uncontaminated type-1 cells. We consider the possible implications of our work for reports of KHE in other cell types.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004240050417
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of acidic stimuli on intracellular calcium in isolated type I cells of the neonatal rat carotid body (1993)
    Springer
    Pflügers Archiv 425 (1993), S. 22-27 
    Details
    ISSN: 1432-2013
    Keywords: Carotid body ; Chemoreceptor ; Intracellular calcium ; Intracellular pH ; Extracellular pH ; Acidosis ; Hypercapnia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have investigated the effects of acidic stimuli upon [Ca2+]i in isolated carotid body type I cells from the neonatal rat using indo-1 (AM-loaded). Under normocapnic, non-hypoxic conditions (23 mM HCO3 −, 5% CO2 in air, pHo=7.4), the mean [Ca2+]i for single cells was 102±5.0 nM (SEM, n=55) with 58% of cells showing sporadic [Ca2+]i fluctuations. A hypercapnic acidosis (increase in CO2 to 10%–20% at constant HCO3 −, pHo 7.15–6.85), an isohydric hypercapnia (increase in CO2 to 10% at constant pHo=7.4) and an isocapnic acidosis (pHo=7.0, constant CO2) all increased [Ca2+]i in single cells and cell clusters. The averaged [Ca2+]i response to both hypercapnic acidosis and isohydric hypercapnia displayed a rapid rise followed by a secondary decline. The averaged [Ca2+]i response to isocapnic acidosis displayed a slower rise and little secondary decline. The rise of [Ca2+]i in response to all the above stimuli can be attributed to no single factor other than to a fall of pHi. The hypercapnia-induced rise of [Ca2+]i was almost completely abolished in Ca2+-free solution, suggesting a role for Ca2+ influx in triggering and/or sustaining the [Ca2+]i response. These results are consistent with a role for type I cell [Ca2+]i in mediating pH/PCO2 chemoreception.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00374499
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  • 4
    Electronic Resource
    Electronic Resource
    Phenytoin-induced connective tissue growth in the rat (1986)
    New York, NY [u.a.] : Wiley-Blackwell
    The @Anatomical Record 215 (1986), S. 99-105 
    Details
    ISSN: 0003-276X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Rats were treated daily for 9 days with 100, 50, or 25 mg/kg phenytoin i.p. This treatment resulted in a significant increase in the thickness of the connective tissue capsules of the liver, spleen, and pancreas, and of the subepithelial connective tissue of the mesentery but not the epicardium or visceral pleura of the lung where exposure to the drug was via the vascular route. Many areas of connective tissue growth exhibited obvious proliferation of fibroblasts and in some areas contained seemingly large numbers of macrophages and an increase in vascularity. It was demonstrated by electron microscopy that the macrophages occasionally were seen in intimate contact with the fibroblasts.Our observations clearly showed that intraperitoneal exposure of visceral connective tissues of the rat to phenytoin rapidly resulted in a dose-related proliferation of that tissue. The presence of numerous macrophages leads to the suggestion that macrophage-derived growth factor could be responsible for the increased growth.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/ar.1092150203
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  • 5
    Electronic Resource
    Electronic Resource
    Intracellular signal transduction mechanisms of rat epididymal spermatozoa and their relationship to motility and metabolism (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 77-84 
    Details
    ISSN: 1040-452X
    Keywords: Cauda epididymidis ; Sperm activation ; Calcium ions ; Guanylate cyclase ; Adenylate cyclase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The role of intracellular signal transduction mechanisms in regulating the motility and metabolism of rat spermatozoa in undiluted caudal epididymal fluid (CEF) was examined. Samples of CEF containing immotile spermatozoa were exposed to drugs and other agents that either stimulate signal transduction pathways or mimic the action of their second messengers. Under these conditions, sperm motility in 25-30 nl of CEF was stimulated by calcium ions (Ca2+), N,2′ -O-dibutyryl-guanosine 3′:5′ -cyclic monophosphate (dibutryl cGMP), cyclic adenosine 3′:5′-monophosphate (cAMP), N6,2′-O-dibutyryladenosine 3′:5′ -cyclic monophosphate (dibutyryl cAMP), 8-bromoadenosine 3′:5′ -cyclic monophosphate (8-bromo cAMP), caffeine, theophylline and bicarbonate ions (HCO3-). Other agents such as magnesium ions (Mg2+), veratridine, phospholipase C (PLC), ionophore A23187, 1,2-dioctenoyl-sn-glycerol (DAG), phorbol 12-myristate 13-acetate, phospholipase A2 (PLA2), arachidonic acid, and melittin did not significantly influence motility. In the presence of radiolabelled energy substrates, untreated (immotile) spermatozoa in samples of CEF utilised D-[U-14C]glucose and [1-14C]acetate as exogenous energy sources for oxidative metabolism. No detectable 14C-lactate was produced, and none of the drugs altered the rate of glycolytic or oxidative metabolism. The findings suggest that the motility of rat caudal epididymal spermatozoa is regulated by Ca2+ and the guanylate cyclase and adenylate cyclase pathways, but not through the PLC and PLA2 pathways. Also, their metabolism of exogenous substrate was uncoupled from the induction of motility, and their oxidative capacity exceeded the rate of flux of glucose-carbon through the glycolytic pathway. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080380113
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  • 6
    Electronic Resource
    Electronic Resource
    Characterization of low Mr Zona Pellucida binding proteins from boar spermatozoa and seminal plasma (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 33 (1992), S. 108-115 
    Details
    ISSN: 1040-452X
    Keywords: Zona binding proteins ; Seminal plasma ; Fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A group of low Mr (16 kDa - 23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract. © 1992 Wiley-Liss, Inc.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080330115
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  • 7
    Electronic Resource
    Electronic Resource
    Antigens on rat spermatozoa with a potential role in fertilization (1990)
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 25 (1990), S. 286-296 
    Details
    ISSN: 1040-452X
    Keywords: Monoclonal antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Eight monoclonal antibodies (McAbs), directed against antigens on rat cauda epididymal spermatozoa, were tested for their capacity to interfere with fertilization in vitro as a means of identifying molecules a potential role in sperm-egg recognition and fusion. Antigens recognized by the McAbs were visualized on live spermatozoa by indirect immunofluorescence (IIF) and characterized by immunoblotting. Five McAbs (designated 1B5, 2C4, 4B5, 5B1, and 8C4) recognized antigens specifically on the sperm acrosome and three (designated 2B1, 2D6, and 6B2) bound to the flagellum. Of the eight McAbs investigated, three (2B1, 2C4, and 6B2) were effective in blocking fertilization in vitro when added as culture supernalants to mixtures of sperm and eggs. McAb 6B2 was inhibitory due to its ability to agglutinate spermatozoa. McAbs 2B1 and 2C4 did not agglutinate capacitated spermatozoa, had no observable effect on motility, and yet blocked fertilization in a dose-dependent manner. McAb 2C4 did not give a reaction on immunoblots, but the 2B1 antigen was identified as an Mr 40 kD glycoprotein. McAb 2B1 appeared to block fertilization at the level of zona binding, whereas the effects of 2C4 were directed more against zone penetration and/or fusion with the vitellus. When sperm-egg complexes were stained with 2C4 or 2B1 McAbs and viewed by IIF, all spermatozoa that were attached to the zona showed fluorescence on the head. These results suggest that different antigens on the rat sperm head participate in different aspects of the fertilization process and that during capacitation there is either exposure of these antigens or else they migrate to their site of action from the flagellum.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/mrd.1080250311
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  • 8
    Electronic Resource
    Electronic Resource
    The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro (1981)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 109 (1981), S. 323-332 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Human leucocytes incubated in tissue culture fluid of low-sodium concentration (2 mM; iso-osmolarity maintained with choline chloride) reached a new equlibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabainsensitive sodium efflux fell by more than 50% and the ouabain-insensitive rate constant increased nearly threefold in the low-sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain-insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low-sodium medium ouabainsensitive potassium influx exceeded ouabain-sensitive sodium efflux and no ouabain-sensitive potassium efflux could be demonstrated. Ouabain-insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low-sodium cells, suggesting reduced activity of a ouabain-insensitive K:K exchange system.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041090216
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  • 9
    Electronic Resource
    Electronic Resource
    Morphology of the epithelium of the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the adult male rabbitResearch supported by USPHS Grants HD-04290 (DWH) and HD-02344 (DWF), by the Ford Foundation (DWF) and by a Milton Fund grant from Harvard University (DWH). (1979)
    New York, NY [u.a.] : Wiley-Blackwell
    American Journal of Anatomy 156 (1979), S. 373-400 
    Details
    ISSN: 0002-9106
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The fine structure of the epithelium lining the extratesticular rete testis, ductuli efferentes and ductus epididymidis of the rabbit has been investigated. In the ductuli efferentes the epithelium is composed of two cell types, principal cells and ciliated cells. The latter cell type is distinguished from principal cells by the presence of cilia projecting into the lumen and the position of the nucleus in the apical half of the cell. Principal cells in this segment are characterized by micropinocytotic vesicles on the surface plasma membrane and a variety of small dense bodies scattered throughout the cytoplasm. In the ductus epididymidis basal cells replace ciliated cells as the second cell type, but differences between various segments of the epididymis are related to the fine structure of the principal cells. In the proximal caput epididymidis (Nicander's region 1) the principal cells are tall with long microvilli. They typically contain a small Golgi apparatus and a cluster of dense bodies adjacent to the nucleus. In the distal caput epididymidis (Nicander's regions 2-5) the apical cytoplasm of principal cells is filled with numerous micropinocytotic vesicles and large multivesicular bodies; these features are interpreted as signs of absorptive activity. The multivesicular bodies are absent from the cytoplasm of principal cells in the corpus epididymidis (Nicander's region 6) and, instead, numerous elements of smooth endoplasmic reticulum, a large Golgi apparatus, lipid droplets and dense bodies characterize principal cells in this segment. Towards the proximal cauda epididymidis (Nicander's region 7), the number of dense bodies (lysosomes) in the cytoplasm increases considerably. In the globose cauda (Nicander's region 8), the principal cells are reduced in height, and in addition to the features described in region 7, are characterized by a concentric array of rough endoplasmic reticulum in the basal cytoplasm. These observations are discussed in relation to the role of the epididymis in promoting the maturation and survival of spermatozoa.
    Additional Material: 28 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/aja.1001560307
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  • 10
    Electronic Resource
    Electronic Resource
    A growth factor from spinal cord (1979)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 100 (1979), S. 273-277 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mitogenic activity is present at a variety of sites in the central nervous system. A growth factor was purified from neonatal bovine spinal cord. It has a pI of 9.5-9.8 and a molecular weight of about 11,000 daltons. Spinal cord growth factor is a basic polypeptide that is inactivated by extremely acid or basic conditions. Its mobility on SDS polyacrylamide gels suggests that this factor is different from pituitary FGF and brain FGF-1.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041000208
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