Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.     

Calcium Channel α2δ Subunits: Differential Expression, Function, and Drug Binding

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.     

Delayed leukocytosis after hard strength and endurance exercise: Aspects of regulatory mechanisms

Background  

During infections, polymorphonuclear neutrophilic granulocytes (PMN) are mobilized from their bone marrow stores, travel with blood to the affected tissue, and kill invading microbes there. The signal(s) from the inflammatory site to the marrow are unknown, even though a number of humoral factors that can mobilize PMN, are well known. We have employed a standardized, non-infectious human model to elucidate relevant PMN mobilizers. Well-trained athletes performed a 60-min strenuous strength workout of leg muscles. Blood samples were drawn before, during and just after exercise, and then repeatedly during the following day. Cortisol, GH, ACTH, complement factors, high-sensitive CRP (muCRP), IL-6, G-CSF, IL-8 (CXCL8) and MIP-1β (CCL4) were measured in blood samples. PMN chemotaxins in test plasma was assessed with a micropore membrane technique.     

Cholecystokinin activates both A- and C-type vagal afferent neurons

Patch-clamp electrophysiological methods were used on dissociated rat nodose neurons maintained in culture to determine whether responses to cholecystokinin (CCK) were associated with capsaicin-resistant (A type) or capsaicin-sensitive (C type) neurons. Nodose neurons were classified as A or C type on the basis of the characteristics of the Na+ current, a hyperpolarization-activated current, and sensitivity to a low concentration of capsaicin to ascertain the presence of vanilloid receptor 1 that has been associated with C-type neurons in sensory ganglia. It was expected that only capsaicin-sensitive C-type neurons would respond to CCK, because most vagally mediated actions of CCK are blocked by capsaicin treatment. However, we found that subpopulations of both A- and C-type neurons responded to CCK (24 and 38%, respectively). Thus some vagally mediated actions of CCK may be mediated by capsaicin insensitive A-type neurons.     

Pyrithiones as antifoulants: Environmental chemistry and preliminary risk assessment

Pyrithiones are widely used as bactericides, fungicides, or algicides in a variety of products such as shampoos, metal working fluids, adhesives, sealants, and coatings. This broad antimicrobial activity, along with low water solubility and favorable environmental chemistry, makes zinc pyrithione and copper pyrithione potentially ideal replacements for TBT in marine antifouling paints. Several studies on the toxicity and environ- ? mental fate of these pyrithiones have been conducted in freshwater and saltwater systems. Environmental fate studies show that pyrithiones rapidly degrade in the water column to less toxic compounds. Sediment accumulation is also prevented by the facile reduction of a critical functional group under anaerobic conditions. Modeling programs were used to calculate the predicted environmental concentration (PEC) for pyrithione. Comparison of PECs calculated for more persistent antifoulants with actual measured concentrations provided a measure of the bias inherent to the models. The results indicate a pyrithione risk quotient (PEC/PNEC) < 1. The findings are consistent with the absence of ecological effects during the long history of the use of zinc pyrithione as an antidandruff agent.     

Adaptation to high-fat diet reduces inhibition of gastric emptying by CCK and intestinal oleate

Rats maintained on low-fat (LF) or high-fat (HF) diets were fitted with gastric cannulas and duodenal catheters. Intraperitoneal injection of 0.250-2.0 microg/kg cholecystokinin (CCK) significantly inhibited gastric emptying of a 5-ml NaCl load in LF rats by 26.2-55. 1% compared with emptying after vehicle injection. By contrast, CCK-induced inhibition of gastric emptying was significantly less in HF rats given the same CCK doses (10.0-31.7% inhibition over the same CCK dose range). A 20-min intraduodenal infusion of oleate (0.03 or 0.06 kcal/ml) also resulted in significant inhibition of gastric emptying in LF rats (24 and 89%, respectively). Oleate-induced inhibition of gastric emptying was significantly attenuated in rats maintained on the HF diet (2 and 56%, respectively). Unlike CCK injections or oleate infusion, intraduodenal maltotriose infusion inhibited gastric emptying to a similar degree in LF and HF rats (77 and 78%, respectively). These results indicate that feeding HF diets diminishes the enterogastric inhibition of gastric emptying by intestinal oleate and diminishes the ability of CCK to inhibit gastric emptying.     

Endogenous ABA maintains shoot growth in tomato independently of effects on plant water balance: evidence for an interaction with ethylene

To examine whether the reduced shoot growth of abscisic acid (ABA)-deficient mutants of tomato is independent of effects on plant water balance, flacca and notabilis were grown under controlled-humidity conditions so that their leaf water potentials were equal to or higher than those of well-watered wild-type plants throughout development. Most parameters of shoot growth remained markedly impaired and root growth was also greatly reduced. Additional experiments with flacca showed that shoot growth substantially recovered when wild-type levels of ABA were restored by treatment with exogenous ABA, even though improvement in leaf water potential was prevented. The ability of applied ABA to increase growth was greatest for leaf expansion, which was restored by 75%. The ethylene evolution rate of growing leaves was doubled in flacca compared to the wild type and treatment with silver thiosulphate to inhibit ethylene action partially restored shoot growth. The results demonstrate that normal levels of endogenous ABA are required to maintain shoot development, particularly leaf expansion, in well-watered tomato plants, independently of effects on plant water balance. The impairment of shoot growth caused by ABA deficiency is at least partly attributable to ethylene.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

FTIR spectroscopy of complexes formed between metarhodopsin II and C-terminal peptides from the G-protein alpha- and gamma-subunits

Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtalpha(340-350) and Gtgamma(60-71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtalpha (K341R, L349A) and non-farnesylated Gtgamma). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.     

Growth, ageing and death of a photoautotrophic plant cell culture

 Batch cultures of photoautotrophic cell suspensions of Chenopodiumrubrum L., growing in an inorganic medium on CO₂ under a daily balanced light–dark regime of 16 : 8 h could be maintained for approximately 100 d without subcultivation. The long-lived cultures showed an initial cell division phase of 4 weeks, followed by a stationary phase of another 4 weeks, after which ageing and progressive cell death reduced the number of living cells and the cultures usually expired after another 3–4 weeks. These developmental phases of the cell culture were characterised with respect to photosynthetic performance, dark respiration, content of phytohormones and capacity of cell division. Cell division of the majority of the cells finished in the G1- or G0-phase of the cell cycle, caused by a pronounced decline in the endogenous levels of auxin and cytokinins. Supply of these growth factors to resting cells resulted in resumption of cytokinesis, at least by some of the cells. However, responsiveness to the phytohomones declined during the stationary phase, and subcultivation was no longer possible beyond day 60 when the phases of ageing and death commenced. Ageing was characterised by a further decline in the photosynthetic capacity of the cells, by a climacteric enhancement of dark respiration, but also by a slight increase in the level of IAA and cytokinins concomitant with a decrease in ethylene. Similarities and differences between the development of batch-cultured photoautotrophic cells of C. rubrum and that of a leaf are discussed with respect to using the cell culture as a model for a leaf. Received: 30 April 1999 / Accepted: 21 August 1999