The localization of [3H]-desipramine in central nerve terminals studied with electron microscope autoradiography and subcellular fractionation.

The cellular and subcellular distribution of [3H]-desipramine (DMI) in rat brain was studied by electron microscope (EM) autoradiography and by subcellular fractionation. A considerable proportion of label was found to be bound to the membranes of presynaptic nerve terminals, as well as to sites inside those terminals.     

Concerning the ionic basis of presynaptic inhibition

Summary The superfused rat cuneate nucleus has been used to investigate the sensitivity of primary afferent terminals and of evoked primary afferent depolarization (PAD) to alterations in extracellular K⁺ and Cl^– ion levels. Results indicate that PAD is caused by an efflux of Cl^– from primary afferent terminals rather than by an increase in extracellular K⁺.     

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

Poly-ADP-ribose polymerases (PARPs) use NAD⁺ as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD⁺ detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD⁺ pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD⁺ and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.     

Effects of a new cholecystokinin antagonist (GE 410) on the smooth muscle of the guinea pig ileum

Summary Suc-Tyr-(SE)-Met-Gly-Trp-Met-Asp--phenethylamide (GE 410) competitively antagonized the contractions of smooth muscle strips from guinea pig ileum (pA₂=7.6, n=0.95) induced by cholecystokinin-octapeptide (CCK8). GE 410 inhibited the electrically-induced cholinergically mediated contractile responses and the [³H]ACh release in the ileum, as well as the CCK-stimulated electrical contractile responses and the [³H]ACh release in the cholinergic nerve terminals. The results suggest the existence of CCK-receptors not only in the smooth muscles but also on the neurons.     

High K+-induced release of somatostatin from the cortical preparation of rat brain

Summary Release of endogenous somatostatin (SRIF) from the rat cerebral cortical slices incubated in Krebs-bicarbonate buffer was increased from the basal rate of 3.4±0.6% of the total SRIF content in 15 min at [K⁺]_o=5.6 mM, to 13.1±1.6% upon raising the [K⁺]_o to 56.6 mM. The high-K⁺ evoked SRIF release was absent when Ca⁺⁺ in the medium was replaced by Mn⁺⁺. The isolated synaptosomes from rat cerebral cortex contain 13.2±3.1 ng SRIF/mg protein compared to 0.33±0.01 ng/mg protein in the cortical tissue as a whole, suggesting that nerve terminals are the main source of the peptide released upon membrane depolarization.The study was supported by a grant from the Medical Research Council of Canada. Results of this work have been published in part as abstracts: Can. Physiol.9, 45 (1978), and Fedn Proc.37, 665 (1978).The authors are greatly indebted to Dr M. Gotz and the Ayerst Research Laboratories for the most generous supply of the synthetic somatostatin.     

Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe

Effects of extracellular magnesium ions ([Mg²⁺]_o ) on intracellular free Mg²⁺ ([Mg²⁺]_i ) and its subcellular distribution in single fission yeast cells, Schizosaccharomyces pombe, were studied with digital-imaging microscopy and an Mg²⁺ fluorescent probe (mag-fura-2). Using 0.44 mM [Mg²⁺]_o , [Mg²⁺]_i in yeast cells was 0.91±0.08 mM. Elevation of [Mg²⁺]_o to 1.97 mM induced rapid (within 5 min) increments in [Mg²⁺]_i (2.18±0.11 mM). Lowering [Mg²⁺]_o to 0.06 mM, however, exerted no significant effects on [Mg²⁺]_i (0.93±0.14 mM), at least for periods of up to 30 min. Irrespective of the [Mg²⁺]_o used, the subcellular distribution of [Mg²⁺]_i remained hetero geneous, i.e. where the sub-plasma membrane region >cytoplasm >nucleus. [Mg²⁺] in all three subcellular compartments increased significantly, two- to threefold, concomitant with [Mg²⁺]_i when placed in 1.97 mM [Mg²⁺]_o . We conclude that [Mg²⁺]_i in fission yeast is maintained at a physiologic level when [Mg²⁺]_o is low, but intracellular free Mg²⁺ rapidly rises when [Mg²⁺]_o is elevated. Like most eukaryotic cells, yeast may have a Mg²⁺ transport system(s) which functions to maintain gradients of Mg²⁺ from the outside to inside the cell and among its subcellular compartments. Received 18 April 1996; received after revision 4 July 1996; accepted 26 July 1996     

Zinc antagonizes the effect of botulinum type A toxin at the mouse neuromuscular junction

Summary Zn²⁺ (10–100 M) elevated the frequency of miniature end-plate potentials (MEPPs) in the mouse diaphragm. The effect did not depend on external Ca²⁺. Botulinum type A toxin (BTX_A, 50 ng/ml) abolished MEPPs almost completely within 30 min. Zn²⁺ (100 M) restored MEPPs and increased their frequency after they had been abolished by BTX_A in Ca²⁺-free solutions. The antagonistic effect of Zn²⁺ in the Ca²⁺-free solution was reduced by exposing the diaphragm to the toxin in the Ca²⁺-free solutions containing high K⁺. Thus, the action of BTX_A is probably enhanced by depolarization of the motor nerve terminals.     

Synaptic vesicle depletion and glutamate uptake in a nerve-muscle preparation of the locust,Locusta migratoria L

Summary Excitatory terminals, depleted of their synaptic vesicles by stimulation at high frequency, were incubated in the radiolabelled putative neurotransmitter, L-glutamate. Quantitative electron microscope autoradiography revealed that the axoplasm of these recovering terminals had accumulated the³H L-glutamate.Acknowledgment. The authors wish to thank Dr N. M. Blackett for running the computer analyses of the autoradiographical results. R. P. Botham gratefully acknowledges the SRC for financial assistance.     

Development of noradrenaline uptake in the human foetal heart

Summary The development of NA-uptake mechanisms in the human foetal heart start at the same time as the adrenergic terminals were visible. The highest³H-NA values in the human foetal heart were only 25–30% of those found in the mouse heart.Acknowledgment. The technical assistance of Miss Marjo Martonen is gratefully acknowledged. This investigation was supported by a grant from the National Research Council for Medical Science, Finland.     

A note on the mechanism of action of UV-irradiation of amphibian embryos

Summary The actions on amphibian embryos of UV-irradiation, exposure to Li⁺ or exposure to ouabain show interesting parallels with their effects on spontaneous release at the presynaptic terminals of the neuromuscular junction. It is suggested that these treatments serve to raise intracellular Ca²⁺ ([Ca²⁺ _i) in these examples, and that UV-promoted abnormalities in embryogenesis are a consequence of changes in [Ca²⁺]_i at critical stages in development.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilation at labor. Incubation of cervical fibroblasts with [³H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [³H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (K _d) of 12 nM, and the binding capacity (B _max) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [³H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

The effect of glutamate,cysteate and related amino acids on insect muscle

Summary Application of Glu, CySO₂ or CySO₃ to blowfly larvae caused paralysis and increased the membrane potential of larval muscle. The contents of Glu and CySO₃ in larval muscle containing motor nerve terminals were markedly decreased after the perfusion of high K⁺ saline solution.     

The biotransformation of tritiated 3-dehydroecdysone by crayfish,Procambarus clarkii

The injection of 3-dehydroecdysone (3dhE, 5 /g), the major ecdysteroid secreted by the Y-organ of crayfishProcambarus clarkii, resulted in apolysis within about 5 days. The hormonal response at the molecular level was investigated by injection of the radio-labeled compound; within 3 h of injection of [³H]3dhE, most radio-isotope was found in the extracted epidermal tissues and identified as ecdysone, 20-hydroxyecdysone (20E), and their 3-hydroxy epimers. The biotransformation was undoubtedly performed in the peripheral area of the Y-organ. Cleavage of the polar conjugates, using an enzyme fromHelix pomatia, gave all of the above ecdysteroids including 3dhE. It was also found that the biosynthetic site of 3dhE was different from that of ecdysone at the subcellular level of the Y-organ.     

Calcium and sodium distribution and movements in smooth muscle

Summary Electron probe microanalysis (EPMA) has been used to study the subcellular distribution of Ca, Na, K. Cl, and Mg in smooth muscle. The EPMA results indicate that the sarcoplasmic reticulum (SR) is the majorintracellular source and sink of activator Ca: norepinephrine decreases the Ca content of the junctional SR in portal vein smooth muscle. Mitochondria do not play a significant role in regulating cytoplasmic free Ca²⁺, but mitochondrial Ca content can be altered to a degree compatible with suggestions that fluctuations in matrix Ca contribute to the control of mitochondrial metabolism. The rise intotal cytoplasmic Ca during a maintained, maximal contraction is very much greater than the rise in free Ca²⁺, and is probably in excess of the known binding sites available on calmodulin and myosin. Cell Ca is not increased in normal cells that are Na-loaded. The non-Donnan distribution of Cl is not due to compartmentalization, but reflects high cytoplasmic Cl. Na-loading of smooth muscle in K-free solutions is temperature dependent, and may exhibit cellular heterogeneity undetected by conventional techniques. The total cell Mg is equivalent to approximately 12 mM, and less than 50% of it can be accounted for by binding to ATP and to actin. Mitochondrial monovalent cations in smooth muscle are relatively rapidly exchangeable.     

A presynaptic effect of whole venom ofDendroaspis jamesoni on the hemidiaphragm of rat

Summary Dendroaspis jamesoni venom in a dose of 12.5 g/ml restricts the uptake of Ca⁺ leading to inhibition of release of Ach from the motor nerve terminals of the hemidiaphragms of rats.Acknowledgment. University of Nairobi, for the research grant (No. 670-052), which supported this work. We also thank Mr. S. K. Githaiga, Government Chemist's Department, for his help in the use of the spectrophotometer.     

Diffusion of d-glucose measured in the cytosol of a single astrocyte

Astrocytes interact with neurons and endothelial cells and may mediate exchange of metabolites between capillaries and nerve terminals. In the present study, we investigated intracellular glucose diffusion in purified astrocytes after local glucose uptake. We used a fluorescence resonance energy transfer (FRET)-based nano sensor to monitor the time dependence of the intracellular glucose concentration at specific positions within the cell. We observed a delay in onset and kinetics in regions away from the glucose uptake compared with the region where we locally super-fused astrocytes with the d-glucose-rich solution. We propose a mathematical model of glucose diffusion in astrocytes. The analysis showed that after gradual uptake of glucose, the locally increased intracellular glucose concentration is rapidly spread throughout the cytosol with an apparent diffusion coefficient (D _app) of (2.38 ± 0.41) × 10^?10 m² s^?1 (at 22–24 °C). Considering that the diffusion coefficient of d-glucose in water is D = 6.7 × 10^?10 m² s^?1 (at 24 °C), D _app determined in astrocytes indicates that the cytosolic tortuosity, which hinders glucose molecules, is approximately three times higher than in aqueous solution. We conclude that the value of D _app for glucose measured in purified rat astrocytes is consistent with the view that cytosolic diffusion may allow glucose and glucose metabolites to traverse from the endothelial cells at the blood–brain barrier to neurons and neighboring astrocytes.     

Mechanical and biochemical characterization of the contraction elicited by a calcium-independent myosin light chain kinase in chemically skinned smooth muscle

Summary The contraction induced by a Ca²⁺-independent myosin light chain kinase (MLCK-) was characterized in terms of isometric force (F_o), immediate elastic recoil (SE), unloaded shortening velocity (V_us), shortening under a constant load and ATPase activity of chemically skinned smooth muscle preparations. These parameters were compared to those measured in a Ca²⁺-induced contraction to assess the nature of cross bridge interaction in the MLCK-induced contraction. F_o developed in chicken gizzard fibers as well as SE were similar in contractions elicited by either agent. V_us in the contraction induced by MLCK-(0.36 mg/ml) was similar though averaged 39.3±8.9% less than V_us induced by Ca²⁺ (1.6x10^–6M) in the control fibers. Addition of Ca²⁺ (1.6x10^–6M) to a contraction induced by MLCK-resulted in small increases in both F_o and V_us. Shortening under a constant load was similar for both types of contractions. The contraction induced by MLCK-was accompanied by an increased rate of ATP hydrolysis. The MLCK-induced contraction is thus kinetically similar though not identical to a contraction induced by Ca²⁺. We conclude that with respect to actin-myosin interaction, MLCK- and Ca²⁺-induced contractions are similar.     

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2

Selective pharmacological Na⁺/H⁺ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit ²²Na⁺ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na⁺-dependent (NHE3-sensitive) component of apical dipeptide ([¹⁴C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical ²²Na⁺ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [¹⁴C]Gly-Sar uptake in the absence of Na⁺ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H⁺ electrochemical gradient). EIPA (at 100 μM) has similar effects, but at higher concentrations (>200 μM) also has direct inhibitory effects on hPepT1.Received 28 February 2005; received after revision 20 April 2005; accepted 20 May 2005     

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (ΔF508) cystic fibrosis transmembrane conductance regulator

The Ca²⁺ ionophore ionomycin induced cytosolic [Ca²⁺]_i elevation as well as strong activation of Cl⁻ efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl⁻ efflux was abolished by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, whereas both activators and inhibitors of phospholipase A₂ had no effect, indicating the involvement of Ca²⁺-dependent Cl^- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A₂. These results indicate that phospholipase A₂ activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR. Received 27 April 1999; received after revision 11 June 1999; accepted 23 July 1999     

Über die Wirkung blutzuckersenkender Sulfonylharnstoffe auf das isolierte Rattenzwerchfell

Summary The action of blood sugar depressing sulfonylureas on glucose and oxygen uptake, as well as on glycogen content and formation of C¹⁴O₂ from uniformly labelled C¹⁴-glucose was investigated in rat hemidiaphragms incubated in phosphate buffer. The following results were obtained: (1) Tolbutamide and Carbutamide increased the glucose uptake. (2) Tolbutamide decreased the glycogen-content. (3) Oxygen uptake as well as formation of C¹⁴O₂ were increased by Tolbutamide. (4) The action of Tolbutamide and insuline was equal with respect to glucose uptake but different as regarding the glycogen content, oxygen uptake and CO₂-formation.It is concluded that sulfonylureas increase glucose oxidation in the rat hemidiaphragm probably without increasing insulin sensitivity. To our knowledge this mechanism of action has hitherto not been described.