The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min-1 mg-1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue. Experimental Physiology (2003) 88.1, 1-6.

Salivary fluid secretion is dependent upon reflex stimuli mediated by autonomic nerves. In order to determine if immunoglobulin A (IgA) and salivary proteins are secreted in the absence of nerve stimulation, small volumes (< 2 µl) of saliva were consecutively collected from the submandibular duct of anaesthetised rats following rest pauses in order to sample the protein contents of the ductal system. Within the first 5 µl of such saliva collected by parasympathetic nerve stimulation, IgA and other salivary proteins reached peak concentrations that were over 20-fold greater than levels in parasympathetically stimulated saliva subsequently collected during a 5 min period of stimulation. Confocal microscopy of TRITC-labelled IgA added to live, acutely isolated submandibular acini indicated that it did not enter the lumina by paracellular leakage. IgG is thought to enter saliva by paracellular leakage but did not accumulate in luminal saliva in the present study. Electrophoresis suggested that the major proteins secreted in the absence of stimulation were the same as those present in subsequently stimulated saliva. It can be concluded that IgA and other major submandibular proteins are secreted into glandular lumina in the absence of nerve stimulation. The functional significance of such unstimulated protein secretion is at present unclear. Experimental Physiology (2003) 88.1, 7-12.

In the present study, we have investigated the viability of using tetrodotoxin (TTX) to induce selective blockade of myelinated fibre conduction in rabbit sural nerve, and explored some aspects of reflexes evoked by non-myelinated sural nerve afferents before and after application of TTX. In rabbits decerebrated under halothane-nitrous oxide anaesthesia, application of 30 nM TTX to the desheathed sural nerve completely blocked Aβ and Aδ waves of the compound action potential evoked by electrical stimulation of the nerve at 95 times threshold. The amplitude of C-fibre volleys evoked by these stimuli was reduced to a mean of 60 % of pre-treatment values. Reflexes evoked in medial gastrocnemius motoneurones by sural nerve stimulation showed corresponding changes after TTX treatment, with activation latency increasing from 5-7 ms in the control state to > 100 ms after TTX application. Temporal summation (wind up) in long latency reflexes (> 100 ms) was significantly enhanced after application of TTX. These data show that low concentrations of TTX can selectively block conduction in rabbit sural nerve A-fibres, providing a method for studying the central actions of non-myelinated C-fibres in isolation. Experimental Physiology (2003) 88.1, 13-18.

Phasic changes in pressure have been reported to occur in the bladder which are not associated with micturition. Spontaneous intravesical pressure changes can be recorded from bladders in vitro or bladders in vivo isolated from the central nervous system suggesting that the bladder itself is capable of autonomous activity. Experiments using isolated cells and muscle strips indicate that the smooth muscle can generate spontaneous activity. Whether this is the origin of phasic changes in the intact organ remains unknown. The present study set out to establish the presence and characteristics of autonomous activity in the isolated guinea pig bladder. Multiple-point motion analysis and concurrent intravesical pressure recording were used to identify and quantify spontaneous and evoked activity. Highly complex autonomous activity was observed in unstimulated bladders. This activity comprised localised micro-contractions in single or multiple discrete regions, waves of activity and micro-stretches. Low-amplitude phasic 'micro-transients' were seen in the intravesical pressure trace in association with micro-contractions. Incremental increases in the intravesical volume recruited additional areas of activity. Atropine and tetrodotoxin had no effect on the micro-transients or micro-contractions. Exposure to the muscarinic agonist arecaidine (10-300 nM) initially increased the incidence of micro-contractions which subsequently became co-ordinated into phasic pressure rises and contraction waves, interspersed with periods of total quiescence. The findings describe the generation and co-ordination of autonomous activity in the bladder wall and also demonstrate complex phasic activity. This approach has shown the importance of assessing the integrative properties of the entire organ in studies of the physiology and patho-physiology of the bladder. Experimental Physiology (2003) 88.1, 19-30.

Biogenesis of mitochondria is happening constantly due to the physiological and developmental situation of a cell. As mitochondrial biogenesis is a complex process producing about 20 % of cellular protein, the expression of the 1000 genes involved is expected to be coordinated and regulated tightly. The variety of physiological stimuli and differentiation states lead to the idea of a complex network connecting many different regulatory pathways. By analysing nuclear encoded mitochondrial genes some of the factors involved in the regulation and coordination of mitochondrial gene expression were identified. These factors include general transcription factors such as Sp1 or YY1, as well as transcription factors specific for mitochondrial genes like the nuclear respiratory factors NRF1 and 2. An important control function linked to the physiological situation of a cell is triggered by hormones such as steroid and thyroid hormones. Even cell type-specific regulatory proteins like the myogenin transcription factor family have a strong influence on some mitochondrial genes in the specific cellular background. The regulatory function of most of these proteins can be modulated and enhanced by the coactivators PGC-1α and β and PRC. Although regulatory pathways have been characterized in more detail in recent years, no regulation mechanism has been shown to work on all analysed mitochondrial genes, and the general concept of mitochondrial regulation still remains unclear. Experimental Physiology (2003) 88.1, 33-40.

Mitochondria are subcellular organelles, devoted mainly to energy production in the form of ATP, that contain their own genetic system. Mitochondrial DNA codifies a small, but essential number of polypeptides of the oxidative phosphorylation system. The mammalian mitochondrial genome is an example of extreme economy showing a compact gene organization. The coding sequences for two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and 13 polypeptides are contiguous and without introns. The tRNAs are regularly interspersed between the rRNA and protein-coding genes, playing a crucial role in RNA maturation from the polycistronic transcripts. A single major non-coding region, called the D-loop region, contains the main regulatory sequences for transcription and replication initiation. This genetic organization has its precise correspondence in the mode of expression and distinctive structural features of the RNAs. The basic mechanisms of mitochondrial DNA transcription and replication and the main cis-acting elements playing a role in both processes have been determined. Many trans-acting factors involved in mitochondrial gene expression, including the RNA and DNA polymerases, have been cloned or identified. However, the regulatory mechanisms participating in mitochondrial gene expression are still poorly understood. The interest to complete this knowledge is increased by the involvement of mitochondria in human diseases, in basic processes such as heat production, Ca2+ homeostasis and apoptosis, and by their potential role in ageing and carcinogenesis. Experimental Physiology (2003) 88.1, 41-56.

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes. In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components. We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment. Experimental Physiology (2003) 88.1, 57-64.

The scientifically novel, but evolutionarily ancient, so-called uncoupling proteins 2 and 3 (UCP2, UCP3) are structually similar to the archetypical uncoupling protein UCP1. A series of suggestions have been forwarded for their physiological function. We discuss systematically here the pros and cons for these suggestions. We conclude that the novel UCPs do not seem to be physiologically relevant uncoupling proteins; the uncoupling property was apparently a late introduction into the subfamily through the evolution of UCP1. Physiological functions ascribed to UCP2 and UCP3 based on their purported uncoupling property may have to be revised (i.e. any type of thermogenesis, including protection against obesity, protection against the formation of reactive oxygen species and thermogenic involvement in the fever response). The presence of a mixed genetic background in most published studies of UCP2 or UCP3 gene-ablated mice also means that data concerning marked differences in diabetes propensity, infection sensitivity and production of reactive oxygen species may require confirmation in backcrossed mice. The increased expression of UCP2 and UCP3 under conditions of increased fatty acid metabolism implies an as yet undefined role in lipid metabolism. Thus, the novel UCPs should probably be considered as mitochondrial carriers, and the challenge now is to identify the transported molecule. Experimental Physiology (2003) 88.1, 65-84.

Apoptosis is an evolutionary-conserved physiological mechanism to remove cells from an organism. Cellular apoptosis is mediated via an intracellular signalling programme that involves a variety of signalling molecules and cellular organelles including caspases, sphingomyelinases, Bcl-2-like proteins and proteins to cleave the DNA and mitochondria. Mitochondria contain several pro-apoptotic molecules that activate cytosolic proteins to execute apoptosis, block anti-apoptotic proteins in the cytosol and directly cleave nuclear DNA. Mitochondria trap these pro-apoptotic proteins and physically separate pro-apoptotic proteins from their cytoplasmic targets. Apoptosis is then initiated by the release of mitochondrial pro-apoptotic proteins into the cytosol. This process seems to be regulated by Bcl-2-like proteins and several ion channels, in particular the permeability transition pore (PTP) that is activated by almost all pro-apoptotic stimuli. Experimental Physiology (2003) 88.1, 85-90.

It has been known for a long time that isolated mitochondria are able to accumulate large amounts of calcium ions. Before the discovery that endoplasmic reticulum (ER) was the main Ca2+-storing cellular organelle, mitochondria were thought to play a major role in cytoplasmic Ca2+ homeostasis (Carafoli, 2002). After IP3 was discovered and it was shown that IP3 receptors were localized in ER membrane and that Ca2+-binding proteins such as calsequestrin could store large amounts of Ca2+ in the ER, the role of mitochondria in the regulation of cytoplasmic Ca2+ was questioned. However, in recent years, mostly due to the development of new methods, there has been increasing evidence that mitochondria could be an important cytoplasmic Ca2+ sink, especially under conditions of a high cellular Ca2+ load. Experimental Physiology (2003) 88.1, 91-97.

Regularly performed exercise in the form of endurance training produces a well-established adaptation in skeletal muscle termed mitochondrial biogenesis. The physiological benefit of this is an enhanced performance of muscle when subject to endurance exercise. This is not only of great advantage for athletic endeavours, but it also clearly improves the quality of life of previously sedentary individuals and those involved in injury rehabilitation. Here we review the molecular basis for mitochondrial biogenesis in muscle, from the initial signals arising in contracting muscle, to the transcription factors involved in mitochondrial and nuclear DNA transcription, as well as the post-translational import mechanisms required for the synthesis of the organelle. We discuss specific protein components associated with reactive oxygen species production, and suggest some questions which remain unanswered with respect to the role of exercise-induced mitochondrial biogenesis in ageing, apoptosis and disease. Experimental Physiology (2003) 88.1, 99-107.

This review explores the current concepts relating the structural and functional modifications of skeletal muscle mitochondria to the molecular mechanisms activated when organisms are exposed to a hypoxic environment. In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas. In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis. The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1α. A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1α as well as MAP kinase and other signalling pathways is debated. The current evidence suggests that mitochondria themselves could be important players in oxygen sensing. Experimental Physiology (2003) 88.1, 109-119.

Thyroid hormone (T3) has a profound effect on mitochondrial biogenesis. T3-regulated gene expression is mediated by thyroid hormone receptor (TR) binding to thyroid hormone response elements (TREs). In concert with the action of various coactivators and corepressors this interaction leads to a modulation of the chromatin structure and subsequently to a modulation of gene expression of adjacent target genes. However, as numerous genes are endogenous regulated by T3 whereas a TRE appears to be absent in their regulatory elements, a TR-independent pathway of T3-mediated gene regulation is likely. In this review, we discuss the direct mechanisms of TR-dependent regulation of gene expression on the nuclear and mitochondrial genome by T3. We also summarise recent observations on an indirect mechanism of T3 action via intermediate factor(s). We discuss the regulation of nuclear respiratory factor 1 (NRF-1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) by T3, suggesting NRF-1 and PGC-1α as attractive candidate factors for an intermediate factor of T3 action in vivo. Experimental Physiology (2003) 88.1, 121-128.

Thyroid hormone (T3) has a profound effect on mitochondrial biogenesis. T3-regulated gene expression is mediated by thyroid hormone receptor (TR) binding to thyroid hormone response elements (TREs). In concert with the action of various coactivators and corepressors this interaction leads to a modulation of the chromatin structure and subsequently to a modulation of gene expression of adjacent target genes. However, as numerous genes are endogenously regulated by T3, and a TRE appears to be absent in their regulatory elements, a TR-independent pathway of T3-mediated gene regulation is likely. In this review, we discuss the direct mechanisms of TR-dependent regulation of gene expression on the nuclear and mitochondrial genome by T3. We also summarise recent observations on an indirect mechanism of T3 action via intermediate factor(s). We discuss the regulation of nuclear respiratory factor 1 (NRF-1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) by T3, suggesting NRF-1 and PGC-1α as attractive candidates for an intermediate factor of T3 action in vivo.

Regulation of energy metabolism is one of the major functions of steroid hormones. In this process, mitochondria, by way of oxidative phosphorylation, play a central role. Depending on the energy needs of the cell, on the tissue, on the developmental stage and on the intensity of the hormonal stimulus, the response can be an activation of pre-existing respiratory chain components, an increased transcription of nuclear-encoded and/or mitochondrial-encoded respiratory chain enzyme (OXPHOS) genes and of biosynthesis of the respective enzyme subunits or, in extreme cases of high energy needs, an increase in the number of mitochondria and mitochondrial DNA content per cell. Some of the hormonally regulated systems involving effects on nuclear and mitochondrial OXPHOS genes are reviewed in this paper. The possible molecular mechanisms of steroid hormone action on nuclear and mitochondrial gene transcription and possible ways of coordination of transcription in these two separate cell compartments involving direct interaction of steroid receptors with hormone response elements in nuclear OXPHOS genes and in mitochondria and induction/activation of nuclear-encoded regulatory factors affecting mitochondrial gene transcription are presented. Experimental Physiology (2003) 88.1, 129-140.

Non-shivering thermogenesis in brown adipose tissue is the main mechanism for thermoregulatory heat production in small mammals and newborns. During cold acclimation the sympathetic innervation triggers the recruitment of brown adipose tissue by hyperplasia, which involves the proliferation and differentiation of precursor cells, and by hypertrophy of mature brown adipocytes. Mitochondrial biogenesis and increased synthesis of the uncoupling protein 1 (UCP-1) are hallmarks of the thermogenic recruitment process. The severalfold increase of mitochondrial protein content during cold acclimation recruits a large capacity for oxidative phosphorylation. However, UCP-1 increases proton leakage across the inner membrane of brown adipocyte mitochondria and thereby dissipates proton motive force as heat instead of ATP synthesis. During recent years considerable progress has been achieved in the analysis of transcriptional mechanisms controlling Ucp1 gene expression. However, so far only little is known about the molecular basis of cold-induced mitochondrial biogenesis in brown adipose tissue. Experimental Physiology (2003) 88.1, 141-148.

The metabolic and functional diversity of animal mitochondria caused by different mitochondrial compositions due to tissue-specific mitochondrial pathways and tissue-specific differences in expression of isoforms of subunits of enzymes participating in oxidative phosphorylation will be reviewed here. Applying the concept of metabolic control analysis, the relevance of this diversity for the explanation of tissue-specific effects observed in mitochondrial diseases with homoplasmic mitochondrial DNA mutations and nuclear-encoded respiratory chain defects is discussed. Experimental Physiology (2003) 88.1, 149-154.

Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. This review gives an overview of the principal clinical phenotypes and the molecular genetic basis of mitochondrial disorders identified so far. Experimental Physiology (2003) 88.1, 155-166.

Radical oxygen species are a byproduct of normal energy metabolism in mitochondria. The short-lived radicals cause damage to their immediate surrounding, i.e. the mitochondria. While most of this damage will be removed by normal mitochondrial turnover, damage to mitochondrial DNA (mtDNA) can persist and may accumulate with age. Recent evidence indicates that mutant mtDNA molecules can accumulate within individual cells, potentially hampering mitochondrial function. Experimental Physiology (2003) 88.1, 167-174.

The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent Km for exogenous ADP (250-350 µM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 µm) is independent of intrinsic MgATPase activity. However, the Km may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum. Experimental Physiology (2003) 88.1, 175-190.