Flow-induced release of adenosine 5'-triphosphate from endothelial cells of the rat mesenteric arterial bed.

Adenosine 5'-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

Flow-induced release of adenosine 5′-triphosphate from endothelial cells of the rat mesenteric arterial bed

Adenosine 5-triphosphate (ATP) was released into the perfusate of rat isolated mesenteric arterial beds during each of two consecutive increases in flow. There was no significant difference between the amounts of ATP released on each occasion. Substance P was also released into the perfusate by increased flow, although its release was more variable. Removal of the endothelium of the mesenteric vessels with sodium deoxycholate led to a significant reduction (74%) in the amount of ATP released compared with the release before the endothelium had been removed. This suggests that the ATP released into the mesenteric arterial perfusate during increased flow arises from endothelial cells.     

Modelling and knowledge transfer in complexity science

I analyse the construction and transfer of models in complexity science. Thereby, I introduce a distinction between (i) vertical model construction, which is based on knowledge about a specific target system, (ii) horizontal model construction, which is based on the alteration of an existing model and therefore does not require any references to a specific target system; and (iii) the transfer of models, which consists of the assignment of an existing model to a new target system. I argue that, in complexity science, all three of those modelling activities take place. Furthermore, I show that these activities can be divided into two general categories: (i) the creation of a repository of models without specific target systems, which have been created by large-scale horizontal construction; and (ii) the transfer of these models to particular target systems in the natural sciences, which can also be followed by an extension of the transferred model through vertical construction of adaptions and additions to its dynamics. I then argue that this interplay of different modelling activities in complexity science provides a mechanism for the transfer of knowledge between different scientific fields. It is also crucial to the interdisciplinary nature of complexity science.     

Nervous control of smooth muscle by transmitters,cotransmitters and modulators

Conclusion The part played by peripheral neuroeffector control mechanisms has been underestimated. These are additional to central and ganglionic control mechanisms and are much more elaborate than originally thought. While the classical view is that the autonomic nervous system consists largely of antagonistic cholinergic and adrenergic nerves, about sixteen putative neurotransmitters have been proposed in autonomic nerves in the past few years, including various monoamines, polypeptides, purines and amino acids. Modulatory transmitter mechanisms have also been recognized, including prejunctional inhibition or enhancement of transmitter release, postjunctional modulation of transmitter action, and the secondary involvement of locally synthesized hormones and prostaglandins. The existence of more than one transmitter substance in some nerves is now widely recognized, and suggestions have been made about the ways that this can lead to differential peripheral control mechanisms at nerve terminals themselves. The cotransmitters always have synergistic actions on postjunctional effector cells, but two different operating mechanisms are postulated. 1) If both substances are stored in the same vesicles (for example, ACh or NA with ATP), release is closely parallel at all impulse frequencies. Upon release, the cotransmitter, in addition to having a direct action on postjunctional cells, may facilitate the action of the other transmitter and/or act as an inhibitor of its release. Differential actions at different impulse frequencies are achieved post-junctionally by ATP and NA acting via EJP-spike and spike-independent mechanisms, respectively. 2) If the two substances are stored in separate vesicle types (for example ACh or NA with some peptides), then differential release is possible at different impulse frequencies; the peptides released at higher frequencies modulate the role of the classical transmitter, by both prejunctional enhancement of its release and post-junctional facilitation of its action.     

Structure and function of the GroE chaperone

The Escherichia coli proteins GroEL and GroES were the first chaperones to be studied in detail and have thus become a role model for assisted protein folding in general. A wealth of both structural and functional data on the GroE system has been accumulated over the past years, enabling us now to understand the basic principles of how this fascinating protein-folding machine accomplishes its task. According to the current model, GroE processes a nonnative polypeptide in a cycle consisting of three steps. First, the polypeptide substrate is captured by GroEL. Upon binding of the co-chaperone GroES and ATP, the substrate is then discharged into a unique microenvironment inside of the chaperone, which promotes productive folding. After hydrolysis of ATP, the polypeptide is released into solution. Moreover, GroE may actively increase the folding efficiency, e.g. by unfolding of misfolded protein molecules. The mechanisms underlying these features, however, are yet not well characterized.     

The copper-transporting capacity of ATP7A mutants associated with Menkes disease is ameliorated by COMMD1 as a result of improved protein expression

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P_1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.     

The ATP synthase (F0-F1) complex in oxidative phosphorylation.

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P(i). The bacterial and mitochondrial H(+)-ATP synthases both consist of a membranous sector, F0, which forms a H(+)-channel, and an extramembranous sector, F1, which is responsible for catalysis. When detached from the membrane, the purified F1 sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H(+)-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Physiological arousal: a role for hypothalamic systems

The lateral hypothalamus (LH) has long been known as a homeostasis center of the brain that modulates feeding behavior, arousal and reward. The hypocretins (Hcrts, also called orexins) and melanin-concentrating hormone (MCH) are neuropeptides produced in two intermingled populations of a few thousand neurons in the LH. The Hcrts have a prominent role in regulating the stability of arousal, since Hcrt system deficiency leads to narcolepsy. MCH is an important modulator of energy balance, as MCH system deficiency in mice leads to leanness and increased metabolism. Recently, MCH has been proposed to modulate rapid eye movement sleep in rodents. In this review, we propose a working model of the cross-talk between Hcrt and MCH circuits that may provide an arousal balance system to regulate complex goal-oriented behaviors.     

Inorganic polyphosphate induces accelerated tube formation of HUVEC endothelial cells

In this study, the effect of inorganic polyphosphate (polyP) on the initial phase of angiogenesis and vascularization was investigated, applying the HUVEC cell tube formation assay. PolyP is a physiological and high energy phosphate polymer which has been proposed to act as a metabolic fuel in the extracellular space with only a comparably low ATP content. The experiments revealed that polyP accelerates tube formation of human umbilical vein endothelial cells (HUVEC), seeded onto a solidified basement membrane extract matrix which contains polyP-metabolizing alkaline phosphatase (ALP) activity. This effect is abolished by co-addition of apyrase, which degrades ATP to AMP and inorganic phosphate. The assumption that ATP, derived from polyP, activates HUVEC cells leading to tube formation was corroborated by experiments showing that addition of polyP to the cells causes a strong rise of ATP level in the culture medium. Finally, we show that at a later stage of cultivation of HUVEC cells, after 3 d, polyP causes a strong enhancement of the expression of the genes encoding for the two major matrix metalloproteinases (MMPs) released by endothelial cells during tube formation, MMP-9 and MMP-2. This stimulatory effect is again abrogated by addition of apyrase together with polyP. From these results, we propose that polyP is involved either directly or indirectly in energy supply, via ALP-mediated transfer of energy-rich phosphate under ATP formation. This ATP is utilized for the activation and oriented migration of endothelial cells and for the matrix organization during the initial phases of tube formation.     

Altersbedingte Abnahme von Kreatinphosphat und Änderungen der Adeninnukleotide in der Skelettmuskulatur von Ratten

Summary The content of phosphocreatine and of the adenin nucleotides, ATP, ADP and AMP in the white skeletal muscle of rats of different ages has been determined.There is an age-dependent relation between the quantity of phosphocreatine and the ratio of ATP/ADP. Young rats contain relatively much phosphocreatine (up to 57%) and a high ATP/ADP ratio (mean 6.7) while old rats show less phosphocreatine (40.8%) and also a low ATP/ADP ratio (mean 2.2).

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M. Ermini dankt der Schweizerischen Akademie der Medizinischen Wissenschaften für eine Verlängerung seines Stipendiums undI. Szelenyi dankt für ein USA Forschungs-Stipendium an das Institut für experimentelle Gerontologie in Basel. Herrn Prof.F. Verzár sind wir für die Leitung dieser und weiterer Arbeiten zu Dank verbunden.     

Nitrate and nitrite transport in bacteria

The topological arrangements of nitrate and nitrite reductases in bacteria necessitate the synthesis of transporter proteins that carry the nitrogen oxyanions across the cytoplasmic membrane. For assimilation of nitrate (and nitrite) there are two types of uptake system known: ABC transporters that are driven by ATP hydrolysis, and secondary transporters reliant on a proton motive force. Proteins homologous to the latter type of transporter are also involved in nitrate and nitrite transport in dissimilatory processes such as denitrification. These proteins belong to the NarK family, which is a branch of the Major Facilitator Superfamily. The mechanism and substrate specificity of transport via these proteins is unknown, but is discussed in the light of sequence analysis of members of the NarK family. A hypothesis for nitrate and nitrite transport is proposed based on the finding that there are two distinct types of NarK.     

Spermatozoa: models for studying regulatory aspects of energy metabolism

Spermatozoa are highly specialized cells, and they offer advantages for studying several basic aspects of metabolic control such as the role of adenosine triphosphate-(ATP)-homeostasis for cell function, the mechanisms of fatigue and metabolic depression, the metabolic channelling through the cytoplasm and the organization and regulation of glycolytic enzymes. Spermatozoa of four species with different reproductive modes are, introduced and the first results are presented: Spermatozoa of the marine wormArenicola marina are well adapted to external fertilization in sea water with fluctuating oxygen tension: they are motile for several hours in oxygen-free sea water, even when the ATP level is dramatically reduced. Anaerobic ATP production occurs by alanine, acetate and propionate fermentation probably by the same pathways known from somatic cells of this species. Under aerobic conditions the phosphagen system might function like a shuttle for energy-rich phosphate from mitochondria to the dynein-ATPases. Storage of turkey and carp spermatozoa for several hours without exogenous substrates and oxygen results in the degradation of phosphocreatine and ATP to inorganic phosphate and adenosine monophosphate (AMP), respectively. Despite low energy charges, stored spermatozoa of both species are capable of progressive movements. In carp spermatozoa fatigue of motility is not accompanied by the dramatic acidosis one discusses as an important effect in muscle fatigue. Energy metabolism of boar spermatozoa is typically based on glycolysis consuming extracellular carbohydrates and producing lactate and protons. The sperm seem to tolerate low intracellular pH (<6.5). The lack of a phosphagen system (no energy shuttle from mitochondria to the distal dynein-ATPases) is probably compensated by a high glycolytic ATP-production in the mitochondria-free piece of the flagellum.     

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.     

Polyvinylchloride (PVC) particles implantation in mouse liver. A technique for experimental study of schistosome eggs-induced liver pathology

Summary An injection of suspended PVC particles in the caecal vein of mice induces a foreign-body portal granuloma reaction in the liver. Plastic casts of the portal system, after PVC particles implantation, show modifications in the portal bed and are compared with plastic casts obtained in mice infested bySchistosoma mansoni. This technique can be useful to study the cellular dynamics of the portal granuloma and can be a model for schistosomal eggs induced liver pathology.This work was supported by an ATP (INSERM) 18.75.41 and a scholarship from the Société d'Hépatologie Expérimentale.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.     

Synergistic effect of acute hypoxia on flow-induced release of ATP from cultured endothelial cells

Human umbilical vein endothelial cells (HUVECs) in primary cultures were perfused under normoxic or hypoxic conditions. These cells were stimulated twice for 3 min by increased flow (from 0.5 to 3.0 ml/min). Under hypoxic conditions the basal release of ATP was the same as under normoxic conditions, but during increased flow the release was greater (0.58±0.07>0.32±0.04 pmoles/ml/10⁶ cells (+78%), for the first period of stimulation; 0.39±0.05>0.22±0.03 pmoles/ml/10⁶ cells (+79%) for the second period). Further experiments with sequential increments in flow rate showed that under both normoxic and hypoxic conditions, a positive correlation existed between ATP release and the rate of flow but there was always more ATP released under hypoxic conditions regardless of the flow rate.HUVECs in secondary culture (second passage) were similarly stimulated. No differences were observed between normoxic and hypoxic conditions. In both cases, the quantity of ATP released during high flow (0.050±0.004 pmoles/ml/10⁶ cells) was significantly smaller than the quantity of ATP released during low flow (0.09±0.01 pmoles/ml/10⁶ cells).To conclude, since hypoxia alone did not affect ATP release, there appears to be a synergistic relationship between increased shear stress and hypoxia in the stimulation of ATP release from HUVECs. Moreover, the release of ATP under these conditions seems to be a property of highly differentiated endothelial cells.     

The ATP synthase (F0−F1) complex in oxidative phosphorylation

The transmembrane electrochemical proton gradient generated by the redox systems of the respiratory chain in mitochondria and aerobic bacteria is utilized by proton translocating ATP synthases to catalyze the synthesis of ATP from ADP and P_i. The bacterial and mitochondrial H⁺-ATP synthases both consist of a membranous sector, F₀, which forms a H⁺-channel, and an extramembranous sector, F₁, which is responsible for catalysis. When detached from the membrane, the purified F₁ sector functions mainly as an ATPase. In chloroplasts, the synthesis of ATP is also driven by a proton motive force, and the enzyme complex responsible for this synthesis is similar to the mitochondrial and bacterial ATP synthases. The synthesis of ATP by H⁺-ATP synthases proceeds without the formation of a phosphorylated enzyme intermediate, and involves co-operative interactions between the catalytic subunits.     

Behavioral and neuronal mechanisms of cricket phonotaxis

Summary The auditory communication of crickets provides a model system for the analysis of the neuronal mechanisms underlying complex behavior. The song of male crickets attracts females. The necessary and sufficient parameter of the song for the female phonotaxis has been determined by a quantified behavioral analysis. Neuronal correlates of this pattern recognition exist in the cricket brain and give rise to a hypothesis on the mechanism of song pattern recognition. Causal relationships between the orientation of a cricket during phonotaxis and the activity of single identified neurons were found by monitoring and deactivating single neurons during behavior. The different roles of various identifield neurons for sound localization have been tested by this method. The plasticity of the auditory system at both the behavioral and at the neuronal level has been studied after ampytation of one ear, and a mechanism for sound localization with only one ear is proposed.     

Untersuchung von Adenosintriphosphorsäure (ATP), Adenosindiphosphorsäure (ADP), Adenosinmonophosphorsäure (AMP) und Inosinsäure (IMP) in ruhenden und kontrahierten Froschrektusmuskeln

Summary A method for the estimation of muscle nucleotides is described. The separation of nucleotides has been accomplished by paper ionophoresis at high voltages, as well as by paper chromatography. Even little changes in the nucleotide content such as the changes in a single muscular contraction can be well observed by this method.A decrease of 0.25 µ M ATP/g/contraction (i.e. about of the normal ATP content) has been observed in a contracted muscle, when compared with the ATP content of the resting muscle.