An acute decrease in TCA cycle intermediates does not affect aerobic energy delivery in contracting rat skeletal muscle

We tested the hypothesis that an acute decrease in muscle TCA cycle intermediates during contraction would compromise aerobic energy delivery. Male Wistar rats were anaesthetized and the gastrocnemius–plantaris–soleus (GPS) muscle complex from one leg was isolated and perfused with a red cell medium containing either saline (Con) or cycloserine (Cyclo; 0.05 mg g⁻¹), an inhibitor of alanine aminotransferase (AAT). After 1 h of perfusion, the GPS muscle was either snap frozen (Con-Rest, n = 11; Cyclo-Rest, n = 9) or stimulated to contract for 10 min (1 Hz, 0.3 ms, 2 V) with blood flow fixed at 30 ml min⁻¹ (100 g)⁻¹ and then snap frozen (Con-Stim, n = 10; Cyclo-Stim, n = 10). Maximal AAT activity was > 80% lower ( P < 0.001) in both Cyclo-treated groups (Rest: 0.61 ± 0.02; Stim: 0.63 ± 0.01 mmol (kg wet wt)⁻¹ min⁻¹; mean ± s.e.m. ) compared to Con (Rest: 3.56 ± 0.16; Stim: 3.92 ± 0.29). The sum of five measured TCAI (ΣTCAI) was reduced by 23% in Cyclo-Rest versus Con-Rest but this was not different ( P = 0.08). However, after 10 min of contraction, the ΣTCAI was 25% lower ( P = 0.006) in Cyclo-Stim compared to Con-Stim (1.88 ± 0.15 versus 2.48 ± 0.11 mmol (kg dry wt)⁻¹). Despite the acute decrease in TCAI after Cyclo treatment, the contraction-induced changes in markers of non-oxidative energy provision (phosphocreatine, ATP and lactate) and the decline in tension after 10 min of stimulation were similar compared to Con. These data do not support the hypothesis that the total muscle concentration of TCAI is causally linked to the rate of mitochondrial respiration during contraction.     

Combined inhibition of nitric oxide and prostaglandins reduces human skeletal muscle blood flow during exercise

The vascular endothelium is an important mediator of tissue vasodilatation, yet the role of the specific substances, nitric oxide (NO) and prostaglandins (PG), in mediating the large increases in muscle perfusion during exercise in humans is unclear. Quadriceps microvascular blood flow was quantified by near infrared spectroscopy and indocyanine green in six healthy humans during dynamic knee extension exercise with and without combined pharmacological inhibition of NO synthase (NOS) and PG by l -NAME and indomethacin, respectively. Microdialysis was applied to determine interstitial release of PG. Compared to control, combined blockade resulted in a 5- to 10-fold lower muscle interstitial PG level. During control incremental knee extension exercise, mean blood flow in the quadriceps muscles rose from 10 ± 0.8 ml (100 ml tissue)⁻¹ min⁻¹ at rest to 124 ± 19, 245 ± 24, 329 ± 24 and 312 ± 25 ml (100 ml tissue)⁻¹ min⁻¹ at 15, 30, 45 and 60 W, respectively. During inhibition of NOS and PG, blood flow was reduced to 8 ± 0.5 ml (100 ml tissue)⁻¹ min⁻¹ at rest, and 100 ± 13, 163 ± 21, 217 ± 23 and 256 ± 28 ml (100 ml tissue)⁻¹ min⁻¹ at 15, 30, 45 and 60 W, respectively ( P < 0.05 vs. control). In conclusion, combined inhibition of NOS and PG reduced muscle blood flow during dynamic exercise in humans. These findings demonstrate an important synergistic role of NO and PG for skeletal muscle vasodilatation and hyperaemia during muscular contraction.     

Ca2+ permeability of the channel pore is not essential for the δ2 glutamate receptor to regulate synaptic plasticity and motor coordination

The δ2 glutamate receptor (GluRδ2) plays a crucial role in cerebellar functions; mice with a disrupted GluR δ 2 gene ( GluR δ 2^−/− ) display impaired synapse formation and abrogated long-term depression (LTD). However, the mechanisms by which GluRδ2 functions have remained unclear. Because a GluRδ2 mutation in lurcher mice causes channel activities characterized by Ca²⁺ permeability, GluRδ2 was previously suggested to serve as a Ca²⁺-permeable channel in Purkinje cells. To test this hypothesis, we introduced a GluR δ 2 transgene, which had a mutation (Gln618Arg) in the putative channel pore, into GluR δ 2^−/− mice. Interestingly, the mutant transgene rescued the major functional and morphological abnormalities of GluR δ 2^−/− Purkinje cells, such as enhanced paired-pulse facilitation, impaired LTD at parallel fibre synapses, and sustained innervation by multiple climbing fibres. These results indicate that the conserved glutamine residue in the channel pore, which is crucial for all Ca²⁺-permeable glutamate receptors, is not essential for the function of GluRδ2.     

Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D

Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca²⁺-induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca²⁺-induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 versus 475 ± 33 nmol (mg protein)⁻¹ for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca²⁺ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca²⁺ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.     

Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell-populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1⁺ (as well as Vδ1⁻Vδ2⁻) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8⁺γ/δ⁺ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.     

Prenatal hypoxia impairs circadian synchronisation and response of the biological clock to light in adult rats

The present study was undertaken to investigate the fate of blood-borne non-esterified fatty acids (NEFA) entering contracting and non-contracting knee extensor muscles of healthy young individuals. [U-¹³C]-palmitate was infused into a forearm vein during 5 h of one-legged knee extensor exercise at 40 % of maximal work capacity and the NEFA kinetics, oxidation and rate of incorporation into intramuscular triacylglycerol (mTAG) were determined for the exercising and the non-exercising legs. During 4 h of one-legged knee extensor exercise, m tag content decreased by 30 % (   P < 0.05  ) in the contracting muscle, whereas it was unchanged in the non-contracting muscle. The uptake of plasma NEFA, as well as the proportion directed towards oxidation, was higher in the exercising compared to the non-exercising leg, whereas the rate of palmitate incorporation into m tag was fourfold lower (0.70 ± 0.14 vs. 0.17 ± 0.04 μmol (g dry wt)⁻¹ h⁻¹; P lt; 0.05), resulting in fractional synthesis rates of 1.0 ± 0.2 and 3.8 ± 0.9 % h⁻¹ ( P lt; 0.01) for the contracting and non-contracting muscle, respectively. These findings demonstrate that mTAG in human skeletal muscle is continuously synthesised and degraded and that the metabolic fate of plasma NEFA entering the muscle is influenced by muscle contraction, so that a higher proportion is directed towards oxidation at the expense of storage in mTAG.     

An association between cutaneous sensitivity to histamine and HLA phenotype

Four hundred and twenty randomly chosen subjects from a normal population were HLA typed and tested for cutaneous sensitivity to histamine by prick testing with 5 concentrations of histamine (10⁻³, 10⁻², 10⁻¹, 1, 10 mg ± ml⁻¹). Positive responses to 10⁻¹ mg ± ml⁻¹ histamine occurred in 41% of the subjects, and particularly those with HLA-B7 (55%) (p < 0.005). It is concluded that genes within the major histocomptability complex influence cutaneous responses to histamine.     

Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle

The mitochondrial theory of ageing proposes that the accumulation of oxidative damage to mitochondria leads to mitochondrial dysfunction and tissue degeneration with age. However, no consensus has emerged regarding the effects of ageing on mitochondrial function, particularly for mitochondrial coupling (P/O). One of the main barriers to a better understanding of the effects of ageing on coupling has been the lack of in vivo approaches to measure P/O. We use optical and magnetic resonance spectroscopy to independently quantify mitochondrial ATP synthesis and O₂ uptake to determine in vivo P/O. Resting ATP demand (equal to ATP synthesis) was lower in the skeletal muscle of 30-month-old C57Bl/6 mice compared to 7-month-old controls (21.9 ± 1.5 versus 13.6 ± 1.7 nmol ATP (g tissue)⁻¹ s⁻¹, P = 0.01). In contrast, there was no difference in the resting rates of O₂ uptake between the groups (5.4 ± 0.6 versus 8.4 ± 1.6 nmol O₂ (g tissue)⁻¹ s⁻¹). These results indicate a nearly 50% reduction in the mitochondrial P/O in the aged animals (2.05 ± 0.07 versus 1.05 ± 0.36, P = 0.02). The higher resting ADP (30.8 ± 6.8 versus 58.0 ± 9.5 μmol g⁻¹, P = 0.05) and decreased energy charge (ATP/ADP) (274 ± 70 versus 84 ± 16, P = 0.03) in the aged mice is consistent with an impairment of oxidative ATP synthesis. Despite the reduced P/O, uncoupling protein 3 protein levels were not different in the muscles of the two groups. These results demonstrate reduced mitochondrial coupling in aged skeletal muscle that alters cellular metabolism and energetics.     

Mechanical load plays little role in contraction-mediated glucose transport in mouse skeletal muscle

The factors responsible for control of glucose transport during exercise are not fully understood. We investigated the role of mechanical load in contraction-mediated glucose transport in an isolated muscle preparation. Mouse extensor digitorum longus muscles were stimulated with repeated contractions for 10 min with or without N -benzyl-p-toluene sulphonamide (BTS, an inhibitor of myosin II ATPase) to block crossbridge activity. BTS inhibited force production during repeated contraction to ∼5% of control. In contrast, BTS had little effect on glucose transport in the basal state (control = 0.55 ± 0.04; BTS = 0.47 ± 0.09 μmol (20 min)⁻¹ ml⁻¹) or after contraction (control = 2.27 ± 0.15; BTS = 2.10 ± 0.16 μmol (20 min)⁻¹ ml⁻¹). BTS did not significantly alter the contraction-mediated changes in high-energy phosphates, glutathione status (a measure of oxidant status) or AMP-activated protein kinase activity. In conclusion, these data show that mechanical load plays little role in contraction-mediated glucose transport. Instead, it is likely that the increased glucose transport during contraction is a consequence of the increase in myoplasmic Ca²⁺ and the subsequent alterations in metabolism, e.g. increased energy turnover and production of reactive oxygen species.     

γδ T cells: firefighters or fire boosters in the front lines of inflammatory responses

Summary:  Intradermal inoculation of cloned self-reactive αβ T cells into the footpads of mice induced cutaneous graft-versus-host disease (GVHD), and after recovery from GVHD, the epidermis became resistant to subsequent attempts to induce GVHD. Resistance to GVHD was not induced in the epidermis of T-cell receptor δ-deficient (TCRδ^−/−) mice that lacked γδ T cells bearing canonical Vγ5/Vδ1⁺γδTCRs, known as dendritic epidermal T cells (DETCs), and resistance was restored by reconstitution of these mutant mice with precursors of Vγ5⁺ DETCs. Pulmonary infection by Cryptococcus neoformans induced an increase of γδ T cells in the lung, and in comparison with wildtype mice, TCRδ^−/− mice eliminated C. neoformans more rapidly and synthesized more interferon-γ in the lung. In the mouse small intestine, the absence of γδ T cells is associated with a reduction in epithelial cell turnover and downregulation of the expression of major histocompatibility complex class II molecules. The protective role of γδ T cells was verified in a dextran sodium sulfate-induced inflammatory bowel disease (IBD) model, whereas in a spontaneous model of IBD, γδ T cells were involved in the exacerbation of colitis in TCRα^−/− mice. Taken together, in addition to the homeostatic regulation of epithelial tissues, γδ T cells appear to play a pivotal role in the modification of inflammatory responses induced in many organs containing epithelia.     

Elevated renal perfusion pressure does not contribute to natriuresis induced by isotonic saline infusion in freely moving dogs

The study was designed to determine to what extent moderate elevation of renal perfusion pressure (RPP) via the mechanism of 'pressure natriuresis' contributes to the natriuresis induced by acute i.v. saline loading. Nine Beagle dogs maintained on ample sodium intake (5.5 mmol (kg body mass)⁻¹ day⁻¹) were chronically equipped with an aortic occluder to servocontrol RPP, a bladder catheter to measure renal function, and catheters for measurement of RPP and mean arterial blood pressure (MABP). A swivel system allowed free movement in the kennel during experiments. Isotonic saline loading (500 ml in 100 min) was studied as follows: with and without servocontrol of RPP, and these two protocols repeated in the presence of angiotensin-converting enzyme inhibition (ACEI, Enalapril, 2 mg (kg body mass)⁻¹). Saline loading increased MABP by about 12 mmHg and sodium excretion from about 28 μmol min⁻¹ up to about 350 μmol min⁻¹. Without ACEI, servocontrol of RPP at 10% below control 24 h MABP slightly delayed the onset of the saline-induced natriuresis, but did not reduce peak sodium excretion or cumulative sodium excretion. The slight delay most probably resulted from pressure-controlled renin release because, with ACEI, servocontrol of RPP did not delay or reduce the saline-induced natriuresis. In conclusion, pressure natriuresis does not contribute to the natriuresis following acute saline loading.     

Theophylline and cAMP inhibit lysophosphatidic acid-induced hyperresponsiveness of bovine tracheal smooth muscle cells

We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)-embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP- and high K⁺ solution-induced gel contraction was significantly augmented. This was not due to the modulation of Ca²⁺ mobilizing properties, since ATP- and high K⁺-induced Ca²⁺ transients were not significantly different between control and LPA-treated BTSMC. Y-27632, an inhibitor of Rho-kinase, suppressed the LPA-induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> 1 μ m ) reversed the LPA-induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 μ m ). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]_i) in BTSMC. Elevation of [cAMP]_i with dibutyryl cAMP or forskolin also reversed the LPA-induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA-induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA-induced, RhoA/Rho-kinase-mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.     

CD3−4−8− Thymocyte Precursors with Interleukin-2 Receptors Differentiate Phenotypically in Coculture with Thymic Stromal Cells

CD3⁻4⁻8⁻ interleukin-2 receptor positive (IL-2R⁺) thymocyte precursors from adult mice were cocultured with thymic stromal cells from syngeneic adult mice. The IL-2R⁺CD3⁻4⁻8⁻ thymocytes were obtained by positive panning of IL-2R⁺ cells followed by either sorting or negative panning of triple negative cells, and they were cocultured with primary or secondary cultures of heterogeneous thymic stromal cells. Phenotypic maturation of these precursor cells was extremely rapid. Within 2½ days significant numbers of CD4⁺8⁺ and CD3⁺4⁺8⁻ cell populations developed, the latter expressing the αβ T-cell receptor (αβ-TCR). Thus heterogeneous stromal cell cultures support the development of IL-2R⁺ precursors and with these methods it will now be possible to isolate the particular stromal cells involved at each stromal-dependent step.     

Relaxation by vasoactive intestinal polypeptide in the gastric fundus of nitric oxide synthase-deficient mice

In many gastrointestinal tissues nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) both play a role as inhibitory non-adrenergic non-cholinergic neurotransmitters. As the mode of interaction between NO and VIP remains controversial, the aim of this study was to investigate the interplay between NO and VIP in the mouse gastric fundus and to evaluate the nitric oxide synthase (NOS) isoform involved in VIP-induced relaxation by using inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) knockout mice. The influence of NOS inhibitors on the relaxant effect of VIP was determined in isolated smooth muscle cells and smooth muscle strips of wild-type and knockout mice. In isolated smooth muscle cells from wild-type, eNOS knockout and nNOS knockout mice, the relaxation induced by VIP (10⁻⁹ m ) was inhibited by approximately 70–95 % by both the non-selective NOS inhibitor N^G -nitro- l -arginine ( l -NA; 10⁻⁴ m ) and the selective inducible NOS inhibitor N -(3-(aminomethyl)-benzyl)acetamidine (1400W; 10⁻⁶ m ). In cells isolated from iNOS knockout mice, VIP still induced full relaxation but it was not influenced by l -NA or 1400W. In smooth muscle strips from wild-type and knockout mice, the concentration-dependent relaxation by VIP (10⁻⁹ to 3 × 10⁻⁷ m ) was not influenced by l -NA or 1400W. These results suggest that the experimental method determines the influence of NOS inhibitors on the relaxant effect of VIP. iNOS, probably induced by the isolation procedure, might be involved in the relaxant effect of VIP in isolated smooth muscle cells but not in classic smooth muscle strips.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

Exercise hyperaemia: magnitude and aspects on regulation in humans

The primary function of the cardiovascular system is to supply oxygen to tissues and organs in the body. When muscles contract the aerobic demands are met by an increase in oxygen delivery both at the systemic and the regional levels, a match that is very close and holds at submaximal exercise and when small muscle group contract also at vigorous intensities. The level of muscle perfusion reached is 250 ml min⁻¹ (100 g)⁻¹ in muscle of sedentary subjects and in endurance-trained athletes 400 ml min⁻¹ (100 g)⁻¹ has been reported. These levels of peak exercise hyperaemia equal what has been observed in other species. One consequence of these high muscle blood flows is that the human heart cannot support an optimal blood flow in whole body exercise (arms and legs combined) and sympathetically mediated vasoconstriction, also in arterioles feeding active limb muscles, contributes to matching peripheral resistance in order to maintain blood pressure. Respiratory muscles appear to have a higher priority for a blood flow than limb and torso muscles. There is no consensus in regard to which locally produced substances elicit the vasodilatation when muscle contracts. In addition to NO, data are presented for various metabolites of arachidonic acid and also on ATP, possibly released from the red cells. Using blockers of nitric oxide synthase ( l -NMMA or l -NAME) and the enzymes producing epoxyeicosatrienoic acid (EET) (sulpaphenozole or tetraetylammonium chloride) or prostaglandins (indomethacin), muscle blood flow may be reduced by up to 25–40%. Evaluating the exact role of ATP has to await further studies in humans and especially the use of specific ATP receptor blockers.     

Defective regulation of contractile function in muscle fibres carrying an E41K β-tropomyosin mutation

A novel E41K β-tropomyosin (β-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K β-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca²⁺ concentration (apparent rate constant of force redevelopment ( k _tr) and unloaded shortening speed ( V ₀)); and (ii) in contraction sensitivity to Ca²⁺ concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca²⁺ sensitizer EMD 57033. In fibres from patients, 30 μ m of EMD 57033 (i) had no effect on speed of contraction ( k _tr and V ₀) at saturated Ca²⁺ concentration but (ii) increased Ca²⁺ sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K β-Tm mutation.     

Developmental regulation of hepatic and renal gluconeogenic enzymes by thyroid hormones in fetal sheep during late gestation

Tissue glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) activities were investigated in sheep fetuses after experimental manipulation of thyroid hormone status. Increments in hepatic and renal G6P and PEPCK activities seen between 127–130 and 140–145 days of gestation (term, 145 ± 2 days) were abolished when the normal prepartum rise in plasma triiodothyronine (T₃), but not cortisol, was prevented by fetal thyroidectomy (TX). At 127–130 days, hepatic and renal G6P, and renal PEPCK, activities were similar in intact and TX fetuses; however, hepatic PEPCK was increased by TX. At 140–145 days, tissue G6P and PEPCK activities in TX fetuses were lower than in intact fetuses. In immature fetuses infused with cortisol (2–3 mg (kg body wt) ⁻¹ day ⁻¹) for five days, hepatic and renal enzyme activities were increased to those seen in mature fetuses near term. After five days of T₃ infusion (8–12 μg (kg body wt) ⁻¹ day ⁻¹), G6P and PEPCK activities in the liver and kidney were greater than in saline-infused fetuses, but only renal G6P and PEPCK increased to the level seen close to term. Therefore, in fetal sheep, thyroid hormones are important for the prepartum rises in G6P and PEPCK activities in the liver and kidney and may mediate, in part, the maturational effects of cortisol.