Dysregulated Cytokine Production in Human Cystic Fibrosis Bronchial Epithelial Cells

Although pulmonary inflammation is an important pathologic event in cystic fibrosis (CF), the relationship between expression of the CF gene and the inflammatory response is unclear. We studied tumor necrosis factor (TNF) and IL-1 stimulated production of IL-6 and IL-8 by CF, corrected CF, and normal human bronchial epithelial cells in culture. During the first 24 hours of TNF stimulation, CF cells produced significantly more IL-8 than normal or corrected CF cells. In the second 24 hours of TNF stimulation, IL-6 and IL-8 generation ceased in normal and corrected CF cells but accelerated in CF cells, resulting in marked IL-6 and IL-8 accumulation in CF cells. Similar results were found when cells were stimulated with IL-1. Finally, when CF cells were grown at 27°C (a culture condition which results in transport of CF transmembrane conductance regulator, CFTR, to the cell membrane and normalization of chloride conductance) TNF-stimulated production of IL-6 and IL-8 reverted to normal. We conclude that dysregulation of cytokine generation by CF bronchial epithelial cells is directly related to expression of mutant CFTR and these observations provide a potential mechanism for persistence of airway inflammation in CF.     

Presynaptic and postsynaptic effects of inhibitory drugs on the crayfish neuromuscular junction

Summary -aminobutyric acid (GABA) and related drugs reduce the size of the excitatory postsynaptic potential (e.p.s.p.) in the crayfish neuromuscular preparation. The effective inhibitory concentrations of these drugs were compared and the sites of action—postsynaptic and presynaptic—were determined.GABA, -amino--hydroxy-butyric acid, guanidino acetic acid, -alanine and others were found to increase the membrane conductance of the muscle fibers. These drugs also shift the membrane potential in the same direction as the inhibitory postsynaptic potential. It is concluded that this group of drugs imitates the postsynaptic action of the inhibitory transmitter substance.-guanidino-propionic acid (GP), -guanidino-butyric acid and others did not affect the membrane conductance of the muscle fibers. These drugs therefore do not inhibit through the conductance type of postsynaptic inhibition.Both groups of drugs mentioned above (GABA and GP) did reduce the amount of transmitter released from the excitatory nerve terminal per stimulus, they thus have a presynaptic inhibitory effect similar to neural inhibition. This was shown by analysis of the quantum content of the excitatory postsynaptic potential: The inhibitory drugs reduced the number of quanta liberated per stimulus, the size of the quantum remaining constant.Although GP has no direct effects on the membrane resistance of the muscle fiber, it reduces the inhibitory postsynaptic potential and diminishes the action of applied GABA on the membrane conductance. GABA and GP seem to compete for the inhibitory receptor site on the muscle membrane.

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Zusammenfassung -amino-Buttersäure (GABA) und verwandte Substanzen verkleinern das postsynaptische erregende Potential (e.p.s.p.) des Nerv-Muskel-Präparates des Krebses. Die wirksamen hemmenden Konzentrationen dieser Drogen wurden verglichen, und es wurde versucht, Art und Ort der Hemmungswirkungen — postsynaptisch oder präsynaptisch — festzustellen.GABA, -amino--hydroxy-Buttersäure, guanidino-Essigsäure, -Alanin und andere erhöhten die Leitfähigkeit der Muskelzellmembran. Bei Gabe dieser Drogen verschiebt sich auch das Membranpotential in derselben Richtung wie das hemmende postsynaptische Potential. Folglich hat diese Gruppe von Drogen dieselbe Wirkung auf die Muskel-Zell-Membran wie der hemmende neurale Überträgerstoff.-guanidino-Propionsäure (GP), -guanidino-Buttersäure und andere Guanidinosäuren haben keinen Einfluß auf die Membranleitfähigkeit der Muskelzellen. Der hemmende Effekt dieser Drogen ist also nicht vom Typ der postsynaptischen Leitfähigkeitshemmung.Beide oben erwähnten Gruppen von Substanzen (GABA und GP) setzen die Menge des pro Reiz von der erregenden Nervenendigung freigesetzten Überträgerstoffes herab, sie hemmen also präsynaptisch ebenso wie die neurale Hemmung. Diese präsynaptische Hemmung wurde bewiesen durch Analyse des Quantengehalts der erregenden postsynaptischen Potentiale: Die hemmenden Drogen verkleinerten die Zahl der pro Reiz ausgeschütteten Quanten, ohne die Größe des Quantums zu verändern.Obgleich GP keine direkten Effekte auf den Membranwiderstand der Muskelfasern hat, verkleinert es das postsynaptische hemmende Potential und setzt die Wirkung von GABA auf die Leitfähigkeit der Muskelzellmembran herab. GABA und GP verdrängen sich gegenseitig am Hemmungs-Receptor der Muskelmembran, so daß GP den Effekt von GABA kompetitiv hemmt.

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This work was supported by the Deutsche Forschungsgemeinschaft.     

A monovalent ion-selective cation current activated by noradrenaline in smooth muscle cells of rabbit ear artery

We have studied chloride influx and efflux in a highly purified preparation of type n cells freshly isolated from adult guinea-pig lung using ³⁶Cl^–. Chloride uptake was time-dependent, saturable (K_m<10 mM) and was inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS; K_i80 M). In the absence of external chloride (substituted by gluconate), ³⁶Cl^– uptake exhibited an overshoot above equilibrium. The rate of ³⁶Cl^– entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. ³⁶Cl^– efflux was stimulated by external bromide > bicarbonate chloride citrate; and was inhibited by proprionate > acetate > oxalate. Although the chloride channel blocker 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, ³⁶Cl^– influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anionexchange pathway for chloride transport in freshly isolated adult type II pneumocytes.     

Interaction of transforming growth factor β1 with human glomerular epithelial cells in culture: opposite effects on synthesis of matrix proteins and on urokinase plasminogen activator

The effect of transforming growth factor- (TGF-) was analyzed on the synthesis of fibronectin, collagen type IV, and urokinase plasminogen activator in human glomerular epithelial cells in culture. An increase in the abundance of specific mRNA was found for collagen type IV and fibronectin. Fibronectin protein synthesis was also increased in TGF- treated cells; most of the de novo synthesized fibronectin was found as an unsoluble protein associated with extracellular matrix. In the same cells the amount of plasminogen activator mRNA was found leading also to a decreased surface expression of urokinase plasminogen activator. The data support the concept that by upregulating matrix protein synthesis and downregulating the plasminogen activator system, TGF- favors the development of sclerosis.Abbreviations FN Fibronectin - GEC Glomerular epithelial cells - TGF- Transforming growth factor - uPA Urokinase-type plasminogen activator     

Early effects of aldosterone on the basolateral potassium conductance of A6 cells

Summary Aldosterone increases the basolateral conductance in target epithelia. The basolateral membrane of tight epithelia contains two different types of K⁺ conductances (G_K), a resting and a volume-activated G_K. We have studied the early effects (at 4 hours) of 500 nmol/l aldosterone on the basolateral membrane G_k of A6 cells (a Xenopus laevis kidney cell line), after the permeabilization of the apical membrane with amphotericin B. In the presence of a 97 to 3 mmol/l apical to basolateral K⁺ gradient, the resting, inward rectifying G_K was similar in control and aldosterone treated cells. In contrast, aldosterone induced a 2-fold increase of the volume-activated quinidine sensitive G_K.     

Molecular and cytogenetic organization of the 5S ribosomal DNA array in chicken (Gallus gallus)

The 5S ribosomal (r) RNA genes encode a small (120-bp) highly-conserved component of the large ribosomal subunit. The objective of the present research was to study the molecular and cytogenetic organization of the chicken 5S rDNA. A predominant 2.2-kb gene (5S) consisting of a coding and intergenic spacer (IGS) region was identified in ten research and commercial populations. A variant gene repeat of 0.6kb (5S) was observed in some of the populations. Genetic linkage analysis and cytogenetic localization by fluorescence in-situ hybridization assigned the 5S rDNA to chromosome 9. The 5S rDNA array was determined to be 80.2±7.0kb upon electrophoretic sizing following EcoRV digestion. Sequence analysis of 5S IGS regions revealed considerable conservation between chicken subspecies (98.4% identity) as well as homology with vertebrate Pol III promoter and regulatory sequence motifs. Minor intraindividual sequence variation within 1000bp of IGS was observed in four cloned Red Jungle Fowl (Gallus gallus gallus) 5S repeats (95.5% identity in this region). Sequence comparisons between IGS regions of 5S and 5S genes indicated two short continuous (>20bp) and many short non-continuous homologous regions as well as other conserved features such as promoter and termination motifs.     

Nachweis von 6 neuen Steroiden im Plasma von menschlichem Placentablut (Nabelsehnurblut)

Zusammenfassung 16-Hydroxyprogesteron, 17-Hydroxyprogesteron, ⁴-Pregnen-20-ol-3-on, ⁴-Pregnen-17, 20-diol-3-on, Adrenosteron und 11-Hydroxyandrostendion wurden in Extrakten von Plasma des menschlichen Placentablutes (Nabelschnurblut) nachgewiesen.     

Molecular Characterization of Complement Components (C3, C4, and Factor B) in Human Saliva

A molecular analysis of complement components (C3, C4, and factor B) in human saliva was performed by SDS-PAGE and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa -chain linked to a 70-kDa chain by disulfide bonds, and C3 levels ranged from 0.52 to 15.0 /g/ml (n = 15). C4 was detected as a triple-chain molecule (98-kDa chain, 73-kDa chain, and 33-kDa chain) linked by disulfide bonds, and C4 levels ranged from 0.086 to 4.8 g/ml. Factor B was detected as a 100-kDa single chain, and factor B levels ranged from 0.042 to 0.62/g/ml. The sizes and subunit structures of the complement components in human saliva were compatible with those reported in human serum. The results of a hemolytic assay indicated that the complement molecules in human saliva were functionally active. These complement components may participate in the local immune and inflammatory responses in the oral cavity.     

Action of a serum protein on muscular contraction

Summary The mechanism of action of a serum protein isolated from human serum was assessed in several experimental preparations including glycerol-treated muscle fibers, rat heart papillary muscle and isolatedin vitro perfused rat heart. The action of the serum protein was studied also on canine and human heart papillary muscles which were made to respond to electrical stimulation with ultrasonication modified epinephrine. In addition the action of the protein on adenosine 5 triphosphate generated precipitation of purified human actomyosin was investigated.The serum protein enhanced and intensified the generation of ATP induced tension in glycerol-extracted muscle fibers. It intensified the developed tension (DT) and increased the rate of development of tension (dT/dt) without influencing the time peak tension (TPT) of capillary muscles from rat, canine and human hearts in response to electrical stimulation. The serum protein increased the force of contraction of the isolatedin vitro perfused rat heart, and accelerated the adenosine 5 triphosphate generated precipitation of purified human heart actomyosin.     

Suppression by interferon-γ of tumor cell-induced increase in mesothelial permeability

The effect of interferon- (IFN-) on the interaction between tumor cells and mesothelial cell layers was studied from the aspect of changes in mesothelial permeability. Mesothelial permeability was assessed as the percentage diffusion of radiolabeled albumin across the mesothelial cell sheets on Matrigel-coated filter cup assemblies. When lined gastric carcinoma cells (KATO-III) were seeded on the confluent mesothelial cell layers, the fine cobblestone appearance of the cell sheet was disrupted and mesothelial permeability significantly increased. The increase in permeability was suppressed by the addition of as little as 1 U/ml of IFN-. The effect of IFN- was observed when either the conditioned medium of tumor cells alone or the IFN--resistant tumor cells, K-562, was placed onto the mesothelium. The cobblestone appearance of the cell sheet was relatively well preserved in the presence of IFN-. In contrast, IFN- did not suppress tumor-induced mesothelial permeability. These results suggest that IFN- has the potential to protect the human mesothelial cell layers against tumor cells.     

Amygdalar inputs to ADH-secreting supraoptic neurones in rats

Summary Effects of amygdala stimulation on the discharge activity of antidromically identified supraoptic neurosecretory neurones were studied in male rats anaesthetized with urethane. Stimulation of the medial and the basal amygdala produced excitation or inhibition of discharge activity both in phasically firing (phasic) and in continuously firing (continuous) neurones. More phasic neurones were excited than were inhibited after medial amygdala stimulation. On the other hand, fewer continuous neurones were excited by stimulation of the either amygdala area than were inhibited. This difference of responsiveness between phasic and continuous neurones is statistically significant. Synaptic inputs to supraoptic neurosecretory neurones after amygdala stimulation were also observed in rats with a lesion of the stria terminalis. Supraoptic nucleus stimulation activated antidromically 14 of the 336 amygdala neurones tested. Since phasic neurones have been identified as ADH-secreting neurones, it is concluded that ADH-secreting neurones in the rat supraoptic nucleus receive predominantly excitatory synaptic inputs from the medial amygdala and these amygdalar synaptic inputs are mediated by pathways which are at least in part monosynaptic and are not included in the stria terminalis.Supported by the grants nos. 56440079, 56121007 and 56770057 from the Ministry of Education, Science and Culture, Japan     

Reflexphotometrische Untersuchungen zur Gelbfärbung des Schädeldaches bei Diabetes mellitus

Zusammenfassung Die farbvalenzmetrische Untersuchung von 34 Diabetikerschädeln ergab beim Vergleich mit 56 Kontrollfällen den gleichen Farbton und die gleiche spektrale Sättigung. Bei den Schädeln der Diabetiker ließ sich jedoch eine verminderte Helligkeit nachweisen. Die Farbänderung ist daher für eine Vermutungs-diagnose Diabetes mellitus mit Recht zu verwenden. Es wird jedoch vorgeschlagen, nicht mehr von einer Gelbfärbung, sondern von einer Dunklerfärbung zu sprechen. Das Ausmaß der dunkleren Färbung ist eindeutig positiv zur Krankheitsdauer korreliert. Frühestens nach 6 Jahre bestehendem Diabetes ist eine signifikante Farbänderung zu erwarten. Die Ursache dieser Farbänderung ist unbekannt. In Modellversuchen mit ikterischen Schädeln konnte gezeigt werden, daß die farbmetrischen Kurven bei in vivo entstandenem Ikterus und bei künstlich durch Gallenflüssigkeit erzeugter Verfärbung prinzipiell gleich verlaufen.

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Reflex photometric studies of the yellow discoloration of the calvaria in diabetes mellitus

Summary The shade of color and the spectral saturation of the calvariae of 34 diabetic patients were the same colorimetrically as those of 56 control cases. The calvariae of the diabetics showed, however, a reduced brightness. Consequently, the color change may rightfully be used for the presumptive diagnosis of diabetes mellitus. It is suggested, however, that one should not refer anymore to a yellow discoloration, but instead to a darker discoloration. The degree of the darker color is clearly correlated directly with the duration of illness. The earliest a significant color change may be expected is after six years of diabetes. The cause of the color change is unknown. In model experiments it could be shown, that the colorimetric curves of calvariae discolored by icterus during life are principally the same as the curves of calvariae discolored artificially with bile.

     

Interleukin-4 and Interferon-γ Inhibit Prostaglandin Production by Interleukin-1β-Stimulated Human Periodontal Ligament Fibroblasts

The purpose of the present study was to investigate the involvement of cyclooxygease-1 (COX-1) and cyclooxygenase-2 (COX-2) in prostaglandin (PG) production by human periodontal ligament (PDL) fibroblasts stimulated with a proinflammatory cytokine, inerleukin-1 (IL-1), and to examine the effect of interleukin-4 (IL-4), a Th2 cytokine, and interferon- (IFN-), a Th1 cytokine, on PG production by the cells. IL-1-stimulated PDL fibroblasts produced prostaglandin E₂ (PGE₂) in a time-dependent manner. Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE₂ production by IL-1-stimulated cells. Northern blot analysis showed that COX-2 mRNA was detected in IL-1-stimulated PDL cells, although not detected in unstimulated cells, while expression of COX-1 mRNA was in the same extent in both the cells. Dexamethasone inhibited COX-2 mRNA expression, COX activity and PGE₂ production in IL-1-stimulated cells. IL-4 and IFN- suppressed PGE₂ production by IL-1-stimulated PDL fibroblasts, but COX activity enhanced by IL-1 treatment was significantly inhibited by IL-4, not by IFN-. Northern blot analysis showed that IL-4 depressed COX-2 mRNA expression with no effect on COX-1 mRNA expression. On the other hand, IFN- had no effect on expression of COX-1 and -2 mRNA. These data suggest that COX-2 is primarily responsible for PGE₂ production by IL-1-stimulated human PDL fibroblasts and that IL-4 inhibited PGE₂ production by IL-1-stimulated PDL fibroblasts through down-regulation of COX-2 expression, while IFN- suppressed the PGE₂ production with no effect on COX-2 expression.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Interspecific genetic complementation analysis of human and sheep fibroblasts withβ-galactosidase deficiency

Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of -galactosidase and -neuraminidase was homologous with any of four -galactosidase-deficient human diseases. Fibroblasts from -glactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for -galactosidase, with 5-bromo-4-chloro-3-indolyl -d-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from -galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from -galactosidase-deficient sheep and fibroblasts from human or feline GM₁, gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM₁, gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the -galactosidase structural gene.     

Integrins and extracellular matrix-proteins in the different components of the Wilms' tumour

The Wilms' tumour (WT) is composed of blastema, epithelium and mesenchyme; the epithelium and possibly also the mesenchyme develop from the blastema, parallel to embryonal development. Since interactions between cell adhesion receptors and extracellular matrix (ECM) proteins play an important role in tissue maturation, we examined the expression of the integrin subunits 1–6, 1 and 4, and of the ECM proteins fibronectin, laminin and collagen I and IV, in 20 frozen WT samples and in 5 fetal and 2 adult kidneys. The integrin and ECM protein distribution in tumour epithelium and mesenchyme showed strong similarities to that in their fetal counterparts, whereas the tumour blastema differed strongly from the fetal blastema. In the WT blastema different components were recognized. Undifferentiated blastema, characterized by expression of 3 and 6 and the virtual absence of ECM proteins. Blastema with epithelial commitment, showing increased expression of 3 and 6 and the appearance of 2 and, as a very early phenomenon, production of laminin. Blastema with mesenchymal commitment, with loss of 3 and 6 and expression of 1, 4 and 5 and presence of ECM proteins. It is speculated that the inability of the (undifferentiated) blastema to produce ECM proteins is related to its relatively high metastatic potential when compared with epithelium and mesenchyme.     

Cytochemischer Nachweis von Katalaseaktivit?t in der Magenschleimhaut

Zusammenfassung Die Schleimhaut des Magens und des ösophagus von Mäusen wurde untersucht. Die Präparate wurden nach Fixierung in 5% igem Glutaraldehyd zum Nachweis der peroxydatischen Aktivität von Katalase in einem alkalischen (pH 9,5) DAB-Medium inkubiert.Katalaseaktivität konnte in allen Zelltypen des Epithels der Magenschleimhaut nachgewiesen werden (Mucoide Drüsenzellen, Hauptzellen, Belegzellen, Nebenzellen), sowie in den Keratinozyten des geschichteten Plattenepithels in Magen und Ösophagus. Die Enzymaktivität ist in der Matrix membranbegrenzter Zellorganellen von runder, ovaler oder stabförmiger Gestalt zu lokalisieren. (Katalase positive Partikel, CPs). Diese Partikel messen zwischen 0.1 und 0,3 m im Durchmesser, sofern ihr Anschnitt rund ist. Stabförmige Organellen können bis 1 m lang sein, wobei die Abmessungen der kurzen Achsen den Durchmessern kreisförmiger Anschnitte entspricht. Das elektronendichte Reaktionsprodukt entspricht in seinem granulären Aufbau der Struktur der mäßig elektronendichten Matrix dieser Organellen, die zu beobachten ist, wenn keine Enzymreaktion durchgeführt wurde. Die Partikel stimmen mit den in der Literatur als Mikroperoxysomen oder Peroxysomenartige Partikel beschriebenen Organellen überein.

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Cytochemical demonstration of catalase activity in the mucosa of the stomach

Summary The mucosa of the stomach and of the esophagus was investigated in mice. After fixation in 5% glutaraldehyde the tissue was incubated in an alkaline (pH 9.5) DAB-medium to demonstrate the peroxidatic activity of catalase.Catalase activity is found in all cell types of the gastric mucosa (surface epithelial cells zymogenic cells, parietal cells, mucous neck cells) and in keratinocytes of the stratified epithelium of the stomach and of the esophagus. The enzymatic activity is localized in the matrices of membrane bound organelles of round, oval or rod-like shape (catalase positive particles, CPs). Round particles measure from 0.1 m to 0.3 m in diameter. Rod-like organelles are up to 1 m long, while their short axes are similar to the mentioned diameters of round CPs. The granular and electron dense reaction product corresponds to the granular structure of a moderately dense matrix material in these organelles, which is observed in specimens which were not reacted for the demonstration of catalase activity. The catalase positive particles described in this paper resemble the microperoxisomes or peroxisome like particles described in the literature.

Diese Untersuchung wurde mit Unterstützung durch die Hochschuljubiläumsstiftung der Stadt Wien durchgeführt.     

Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K⁺ glutamate-based solution containing 50 nM free Ca²⁺ and 2 mM adenosine 5-triphosphate, dipotassium salt (K₂ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca²⁺ concentrations ([Ca²⁺]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca²⁺] (3–100 M), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200–400 vesicles per cell. Pretreatment of cells with tyramine (10 M for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca²⁺] in the range of 0.1–3 M, the activation rate of secretion increased from 2.3 to 35.3 nA · s^–1, reached a plateau between 3–30 M and rose again from 100 to 1000 M [Ca²⁺] to a maximum of 91.9 nA · s^–1. In contrast, total secretion first increased (0.1–1 M Ca²⁺), then plateaud (1–100 M Ca²⁺) and subsequently decreased (100–1000 M Ca²⁺). At 30 and 1000 M extracellular [Ca²⁺] or [Ca²⁺]_o, the activation rates of secretion from intact cells depolarised with 70 mM K⁺were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 M Ca²⁺) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 A · s at 1000 M Ca²⁺) than in electroporated cells (42.4 A,s at 1 M Ca²⁺). Separation of the time-courses of changes in intracellular [Ca²⁺] or [Ca²⁺]_i and secretion in intact chromaffin cells depolarised with 70 mM K⁺was demonstrated at different [Ca²⁺]_o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca²⁺]_i. In contrast, the decline of secretion was faster than the decline of the peak [Ca²⁺]_i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca²⁺ by the rates of opening and closing of voltage-dependent Ca²⁺ channels of the L-and Q-subtypes, which control the local [Ca²⁺]_i near to exocytotic sites.     

Isoforms of the CD45 common leukocyte antigen family: Markers for human T-cell differentiation

The diverse host defense and immunoregulatory functions of human T cells are performed by phenotypically heterogeneous subpopulations. Among the membrane antigens that are differentially expressed by reciprocal human T-cell subsets are the CD45RA and CD45RO isoforms of the common leukocyte antigen family, which have been hypothesized to identify naive and memory T cells, respectively. The CD45RA antigen is first expressed by T-lineage cells relatively late during their intrathymic maturation and continues to be expressed by most T cells in the immunologically naive neonate. With increasing age and antigenic exposure, however, CD45RA-/RO+ cells become more prevalent in the circulation and comprise the majority of cells in tissues. Analyses of the functional capabilities of CD4+CD45RA+ and CD4+CD45RO+ cells have shown that proliferative responses to memory recall antigens or the ability to provide help for antibody production are functions uniquely performed by CD4+CD45RA-/RO+ cells. The major immunoregulatory functions described for CD4+CD45RA+ cells involve suppression of immune responses, either directly or via the induction of suppressor activity by CD8+ cells. Two general models of differentiation have been proposed to describe the lineal relationship of these T-cell subsets. Although these subsets could represent mature, phenotypically and functionally stable progeny arising from separate differentiation pathways, there is considerable experimental support for the hypothesis that CD45RA-/RO+ cells are memory cells that derive from naive or virgin CD45RA+/RO-precursors via an activation-dependent postthymic differentiation pathway. Altered frequencies of CD45RA+ and CD45RO+ T cells have been observed in a variety of different clinical conditions, particularly diseases manifesting altered immune function. These findings have contributed new information concerning the physiological events regulating thein vivo generation of these T-cell subsets. In addition, they may provide clues to the pathogenetic processes associated with certain diseases.     

Tetrodotoxin exerts a large frequency-dependent depression of the maximum rate of rise of action potentials in guinea pig ventricular myocytes

The action potential configuration in guinea pig ventricular myocytes was unaffected by low concentrations (0.3–1 M) of tetrodotoxin (TTX); high concentrations (10–30 M) depressed both the overshoot (5–10 mV) and duration (5–10%). Although the control was unaffected by stimulation rate (0.1–5 Hz), the depression of by TTX was greatly potentiated at rates above 1 Hz: on dose-response curves, 50% control occurred at 4.3 M (5 Hz) versus 22 M ( 1 Hz). The frequency dependent component of the depression reported here is much larger than the extra block of Na channels observed by others in voltage clamp studies on Purkinje strands. This is not a discrepancy; rather it is a consequence of a non-linear relation between and available Na conductance.