Fishmeal, plant protein, and fish oil substitution with single-cell ingredients in organic feeds for European sea bass (Dicentrarchus labrax).

Single-cell ingredients (SCI) are considered promising nutrient sources which are produced using environmentally friendly biotechnological processes. The aim of the current study was to evaluate the replacement of fishmeal, plant protein sources, and fish oil with SCI in organic feeds for European sea bass (Dicentrarchus labrax). Bacterial protein, yeast protein, and microalgae were used to replace fishmeal trimmings, soya bean meal, and fish oil from trimmings. Triplicate groups (30 fish per replicate) of European sea bass (14.4 ± 2.4 g) were fed the experimental diets for 71 days. The results showed that the incorporation of SCI at all levels of inclusion significantly enhanced nutrient digestibility. Additionally, growth performance parameters were not affected by SCI inclusion, exhibiting similar or improved values. Moreover, a tendency for improved anterior and posterior gut structure was observed and a significant increase of lysozyme activity at the two highest inclusion levels of SCI was determined. Overall, the results showed that the inclusion of SCI at 15% (bacterial: yeast: algae-9.4: 4.7: 1) is possible without compromising any of the parameters evaluated. According to these findings, a higher substitution of fishmeal trimmings, plant protein sources, and fish oil from trimmings with SCI in organic diets for European sea bass (D. labrax) can be further evaluated in future studies.

Deletion of Sod1 in Motor Neurons Exacerbates Age-Related Changes in Axons and Neuromuscular Junctions in Mice.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Skeletal muscle transcriptomics identifies common pathways in nerve crush injury and ageing.

Motor unit remodelling involving repeated denervation and re-innervation occurs throughout life. The efficiency of this process declines with age contributing to neuromuscular deficits. This study investigated differentially expressed genes (DEG) in muscle following peroneal nerve crush to model motor unit remodelling in C57BL/6 J mice. Muscle RNA was isolated at 3 days post-crush, RNA libraries were generated using poly-A selection, sequenced and analysed using gene ontology and pathway tools. Three hundred thirty-four DEG were found in quiescent muscle from (26mnth) old compared with (4-6mnth) adult mice and these same DEG were present in muscle from adult mice following nerve crush. Peroneal crush induced 7133 DEG in muscles of adult and 699 DEG in muscles from old mice, although only one DEG (ZCCHC17) was found when directly comparing nerve-crushed muscles from old and adult mice. This analysis revealed key differences in muscle responses which may underlie the diminished ability of old mice to repair following nerve injury.

HyPer2 imaging reveals temporal and heterogeneous hydrogen peroxide changes in denervated and aged skeletal muscle fibers in vivo.

To determine the role of denervation and motor unit turnover in the age-related increase in skeletal muscle oxidative stress, the hydrogen peroxide (H2O2) specific, genetically-encoded, fluorescent cyto-HyPer2 probe was expressed in mouse anterior tibialis (AT) muscle and compared with ex vivo measurements of mitochondrial oxidant generation. Crush of the peroneal nerve induced increased mitochondrial peroxide generation, measured in permeabilised AT fibers ex vivo and intra vital confocal microscopy of cyto-HyPer2 fluorescence showed increased cytosolic H2O2 in a sub-set (~24%) of individual fibers associated with onset of fiber atrophy. In comparison, mitochondrial peroxide generation was also increased in resting muscle from old (26 month) mice compared with adult (6-8 month) mice, but no age effect on fiber cytosolic H2O2 in vivo was seen. Thus ageing is associated with an increased ability of muscle fibers to maintain cytosolic redox homeostasis in the presence of denervation-induced increase in mitochondrial peroxide generation.

Redefining the major contributors to superoxide production in contracting skeletal muscle. The role of NAD(P)H oxidases.

The production of reactive oxygen and nitrogen species (RONS) by skeletal muscle is important as it (i) underlies oxidative damage in many degenerative muscle pathologies and (ii) plays multiple regulatory roles by fulfilling important cellular functions. Superoxide and nitric oxide (NO) are the primary radical species produced by skeletal muscle and studies in the early 1980s demonstrated that their generation is augmented during contractile activity. Over the past 30 years considerable research has been undertaken to identify the major sites that contribute to the increased rate of RONS generation in response to contractions. It is widely accepted that NO is regulated by the nitric oxide synthases, however the sites that modulate changes in superoxide during exercise remain unclear. Despite the initial indications that the mitochondrial electron transport chain was the predominant source of superoxide during activity, with the development of analytical methods a number of alternative potential sites have been identified including the NAD(P)H oxidases, xanthine oxidase, cyclooxygenases, and lipoxygenases linked to the activity of the phospholipase A2 enzymes. In the present review we outline the subcellular sites that modulate intracellular changes in superoxide in skeletal muscle and based on the available experimental evidence in the literature we conclude that the NAD(P)H oxidases are likely to be the major superoxide generating sources in contracting skeletal muscle.

Sudden Bilateral Sensorineural Hearing Loss Associated with HLA A1-B8-DR3 Haplotype.

Sudden sensorineural hearing loss may be present as a symptom in systemic autoimmune diseases or may occur as a primary disorder without another organ involvement (autoimmune inner ear disease). The diagnosis of autoimmune inner ear disease is still predicated on clinical features, and to date specific diagnostic tests are not available. We report a case of bilateral sudden hearing loss, tinnitus, intense rotatory vertigo, and nausea in a female patient in which the clinical manifestations, in addition to raised levels of circulating immune complexes, antithyroglobulin antibodies, and the presence of the HLA A1-B8-DR3 haplotype, allowed us to hypothesize an autoimmune inner ear disease. Cyclosporine-A immunosuppressive treatment in addition to steroids helped in hearing recovery that occurred progressively with normalization of the hearing function after a five-month treatment. Cyclosporine-A could be proposed as a therapeutic option in case of autoimmune inner ear disease allowing the suspension of corticosteroids that, at high dose, expose patients to potentially serious adverse events.

Pathogenesis of FOLFOX induced sinusoidal obstruction syndrome in a murine chemotherapy model.

BACKGROUND & AIMS: Sinusoidal obstruction syndrome (SOS) following oxaliplatin based chemotherapy can have a significant impact on post-operative outcome following resection of colorectal liver metastases. To date no relevant experimental models of oxaliplatin induced SOS have been described. The aim of this project was to establish a rodent model which could be utilised to investigate mechanisms underlying SOS to aid the development of therapeutic strategies. METHODS: C57Bl/6 mice, maintained on a purified diet, were treated with intra-peritoneal FOLFOX (n=10), or vehicle (n=10), weekly for five weeks and culled one week following final treatment. Sections of the liver and spleen were fixed in formalin and paraffin embedded for histological analysis. The role of oxidative stress on experimental-induced SOS was determined by dietary supplementation with butylated hydroxyanisole and N-acetylcysteine. RESULTS: FOLFOX treatment was associated with the development of sinusoidal dilatation and hepatocyte atrophy on H&E stained sections of the liver in keeping with SOS. Immunohistochemistry for p21 demonstrated the presence of replicative senescence within the sinusoidal endothelium. FOLFOX induced endothelial damage leads to a pro-thrombotic state within the liver associated with upregulation of PAI-1 (p<0.001), vWF (p<0.01) and Factor X (p<0.001), which may contribute to the propagation of liver injury. Dietary supplementation with the antioxidant BHA prevented the development of significant SOS. CONCLUSIONS: We have developed the first reproducible model of chemotherapy induced SOS that reflects the pathogenesis of this disease in patients. It appears that the use of antioxidants alongside oxaliplatin based chemotherapy may be of value in preventing the development of SOS in patients with colorectal liver metastases.

Regional differences in the modulatory role of the epithelium in sheep airway.

1. The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium-intact or -denuded, first- to fourth-order bronchi to acetylcholine (ACh). 2. We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin-related drugs in epithelium-intact and epithelium-denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH-diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders. 3. There was no difference in the response of first- to fourth-order epithelium-intact bronchi to ACh (1 nmol/L-100 mmol/L) or KCl (5-100 mmol/L). Removal of the epithelium had no effect on ACh-induced contractions of first- and second-order bronchi, but increased responses of third- and fourth-order bronchi to ACh. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) increased ACh-induced contractions of fourth-order epithelium-intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L-1 mmol/L) equally relaxed 1 micromol/L carbachol-precontracted epithelium-denuded first- and fourth-order bronchi. 4. Although NAPDH-diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5-fold greater in fourth- compared with second-order epithelium-intact bronchi. 5. The cyclo-oxygenase inhibitor indomethacin (10 micromol/L) had no effect on ACh-induced contractions of first- to fourth-order epithelium-intact bronchi, but decreased responses of fourth-order epithelium-denuded bronchi to ACh. The contractile effect of the thromboxane A(2) mimetic U-46619 (1 nmol/L-10 micromol/L) was greater in fourth- compared with first-order epithelium-denuded bronchi. 6. In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

Fentanyl treatment reduces GABAergic inhibition in the CA1 area of the hippocampus 24 h after acute exposure to the drug.

The effect of in vivo fentanyl treatment on synaptic transmission was studied in the CA1 area of the rat hippocampus. Animals were treated either with saline or fentanyl (4 x 80 microg/kg, s.c./15 min). Intracellular in vitro recordings were obtained, 24 h after treatment, from CA1 pyramidal neurons. No difference in pyramidal neuron basic membrane properties or postsynaptic membrane excitability was observed between neurons from saline- and fentanyl-treated animals. The peak amplitude of fast (f-) and slow (s-) components of IPSPs elicited in standard ACSF and the peak amplitude and rate of rise of isolated f- and s-IPSPs elicited in the presence of antagonists (CNQX, 10 microM; AP-5, 10 microM; CGP 55845, 1 microM; and bicuculline methochloride, 10 microM), in response to various stimulus intensities, was smaller in fentanyl-treated animals. Conversely, the rising slope of excitatory responses was similar in neurons from saline- and fentanyl-treated animals. Furthermore, in fentanyl-treated animals, lower stimulus strengths were required to elicit subthreshold excitatory responses of the same amplitude suggesting that acute exposure to fentanyl increases susceptibility of pyramidal neurons to presynaptic stimulation. GABA immunohistochemistry revealed lower GABA content in processes and neuronal somata suggesting diminished GABA release onto pyramidal neurons. We conclude that acute in vivo exposure to fentanyl is sufficient to induce long-lasting reduction in GABA-mediated transmission, rather, than enhanced excitatory transmission or modulation of the intrinsic excitability of pyramidal neurons. These findings provide evidence regarding the mechanisms involved in the early stages of tolerance development towards the analgesic effects of opioids.

Genetic modification of the manganese superoxide dismutase/glutathione peroxidase 1 pathway influences intracellular ROS generation in quiescent, but not contracting, skeletal muscle cells.

Increased amounts of reactive oxygen species (ROS) are generated by skeletal muscle during contractile activity, but their intracellular source is unclear. The oxidation of 2',7'-dichlorodihydrofluorescein (DCFH) was examined as an intracellular probe for reactive oxygen species in skeletal muscle myotubes derived from muscles of wild-type mice and mice that were heterozygous knockout for manganese superoxide dismutase (Sod2(+/-)), homozygous knockout for glutathione peroxidase 1 (GPx1(-/-)), or MnSOD transgenic overexpressors (Sod2-Tg). Myoblasts were stimulated to fuse and loaded with DCFH 5-7 days later. Intracellular DCF epifluorescence was measured and myotubes were electrically stimulated to contract for 15 min. Quiescent myotubes with decreased MnSOD or GPx1 showed a significant increase in the rate of DCFH oxidation whereas those with increased MnSOD did not differ from wild type. Following contractions, myotubes from all groups showed an equivalent increase in DCF fluorescence. Thus the oxidation of DCFH in quiescent skeletal muscle myotubes is influenced by the content of enzymes that regulate mitochondrial superoxide and hydrogen peroxide content. In contrast, the increase in DCFH oxidation following contractions was unaffected by reduced or enhanced MnSOD or absent GPx1, indicating that reactive oxygen species produced by contractions were predominantly generated by nonmitochondrial sources.

Somatostatin receptors in wildtype and somatostatin deficient mice and their involvement in nitric oxide physiology in the retina.

The present study investigated the localization and density of somatostatin (SRIF) receptor subtypes (sst(1-5)) and SRIF-nitric oxide (NO()) interactions in the retina of wildtype [WT, (+/+)] and somatostatin deficient mice [SRIF (-/-)]. Immunohistochemistry and radioligand binding studies with subsequent autoradiography were performed. Monoclonal antibodies [SRIF, protein kinase C (rod bipolar cells marker), microtubule associated protein 1A (ganglion cell marker)] and polyclonal antibodies (anti-sst(1), sst(2A), sst(4) receptor) were applied to 10-14 microm sections of retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. [(125)I]LTT SRIF-28 alone or in the presence of MK678 (sst(2) agonist) and [(125)I]Tyr(3)-octreotide were employed to quantify sst(1-5), sst(1/4)and sst(2/5) receptor densities, respectively. sst(1), sst(2A), and sst(4) receptor immunoreactivities were observed in processes of the inner plexiform layer (IPL), rod bipolar, and in ganglion cells and processes, respectively, in WT and SRIF (-/-) mice. Specific [(125)I]LTT SRIF-28 and [(125)I]Tyr(3)-octreotide binding was increased significantly in SRIF (-/-) mice. NADPH-diaphorase staining was localized in photoreceptors and amacrine cells, but not rod bipolar and ganglion cells. Also, NADPH-diaphorase staining was not colocalized with sst(1), sst(2A) or sst(4) receptor immunoreactivity. These results demonstrate an upregulation of SRIF receptors in mice lacking SRIF, but no evident SRIF-NO(*) interaction was observed in the mouse retina.

Adaptive responses of mouse skeletal muscle to contractile activity: The effect of age.

This study has characterised the time course of two major transcriptional adaptive responses to exercise (changes in antioxidant defence enzyme activity and heat shock protein (HSP) content) in muscles of adult and old male mice following isometric contractions and has examined the mechanisms involved in the age-related reduction in transcription factor activation. Muscles of B6XSJL mice were subjected to isometric contractions and analysed for antioxidant defence enzyme activities, heat shock protein content and transcription factor DNA binding activity. Data demonstrated a significant increase in superoxide dismutase (SOD) and catalase activity and HSP content of muscles of adult mice following contractile activity which was associated with increased activation of the transcription factors, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and heat shock factor (HSF) following contractions. Significant increases in SOD and catalase activity and heat shock cognate (HSC70) content were seen in quiescent muscles of old mice. The increase in antioxidant defence enzyme activity following contractile activity seen in muscles of adult mice was not seen in muscles of old mice and this was associated with a failure to fully activate NF-kappaB and AP-1 following contractions. In contrast, although the production of HSPs was also reduced in muscles of old mice following contractile activity compared with muscles of adult mice following contractions, this was not due to a gross reduction in the DNA binding activity of HSF.

Free radical generation by skeletal muscle of adult and old mice: effect of contractile activity.

Oxidative modification of cellular components may contribute to tissue dysfunction during aging. In skeletal muscle, contractile activity increases the generation of reactive oxygen and nitrogen species (ROS). The question of whether contraction-induced ROS generation is further increased in skeletal muscle of the elderly is important since this influences recommendations on their exercise participation. Three different approaches were used to examine whether aging influences contraction-induced ROS generation. Hind limb muscles of adult and old mice underwent a 15-min period of isometric contractions and we examined ROS generation by isolated skeletal muscle mitochondria, ROS release into the muscle extracellular fluid using microdialysis techniques, and the muscle glutathione and protein thiol contents. Resting skeletal muscle of old mice compared with adult mice showed increased ROS release from isolated mitochondria, but no changes in the extracellular levels of superoxide, nitric oxide, hydrogen peroxide, hydroxyl radical activity or muscle glutathione and protein thiol contents. Skeletal muscle mitochondria isolated from both adult and old mice after contractile activity showed significant increases in hydrogen peroxide release compared with pre-contraction values. Contractions increased extracellular hydroxyl radical activity in adult and old mice, but had no significant effect on extracellular hydrogen peroxide or nitric oxide in either group. In adult mice only, contractile activity increased the skeletal muscle release of superoxide. A similar decrease in muscle glutathione and protein thiol contents was seen in adult and old mice following contractions. Thus, contractile activity increased skeletal muscle ROS generation in both adult and old mice with no evidence for an age-related exacerbation of ROS generation.

Preconditioning of skeletal muscle against contraction-induced damage: the role of adaptations to oxidants in mice.

Adaptations of skeletal muscle following exercise are accompanied by changes in gene expression, which can result in protection against subsequent potentially damaging exercise. One cellular signal activating these adaptations may be an increased production of reactive oxygen and nitrogen species (ROS). The aim of this study was to examine the effect of a short period of non-damaging contractions on the subsequent susceptibility of muscle to contraction-induced damage and to examine the changes in gene expression that occur following the initial contraction protocol. Comparisons with changes in gene expression in cultured myotubes following treatment with a non-damaging concentration of hydrogen peroxide (H(2)O(2)) were used to identify redox-sensitive genes whose expression may be modified by the increased ROS production during contractions. Hindlimb muscles of mice were subjected to a preconditioning, non-damaging isometric contraction protocol in vivo. After 4 or 12 h, extensor digitorum longus (EDL) and soleus muscles were removed and subjected to a (normally) damaging contraction protocol in vitro. Muscles were also analysed for changes in gene expression induced by the preconditioning protocol using cDNA expression techniques. In a parallel study, C(2)C(12) myotubes were treated with a non-damaging concentration (100 microM) of H(2)O(2) and, at 4 and 12 h following treatment, myotubes were treated with a damaging concentration of H(2)O(2) (2 mM). Myotubes were analysed for changes in gene expression at 4 h following treatment with 100 microM H(2)O(2) alone. Data demonstrate that a prior period of non-damaging contractile activity resulted in significant protection of EDL and soleus muscles against a normally damaging contraction protocol 4 h later. This protection was associated with significant changes in gene expression. Prior treatment of myotubes with a non-damaging concentration of H(2)O(2) also resulted in significant protection against a damaging treatment, 4 and 12 h later. Comparison of changes in gene expression in both studies identified haem oxygenase-1 as the sole gene showing increased expression during adaptation in both instances suggesting that activation of this gene results from the increased ROS production during contractile activity and that it may play a role in protection of muscle cells against subsequent exposure to damaging activity.

Vitamin E and the oxidative stress of exercise.

There is clear evidence that contracting skeletal muscle generates a complex set of reactive oxygen and nitrogen species and that the pattern and magnitude of this generation is influenced by the type and frequency of the muscle contraction protocol. The functions of these species in exercising organisms are still unclear although data have been presented indicating that they play a role in contraction-induced muscle damage and/or in signaling adaptive responses to contractions. Vitamin E has been claimed to exert a regulatory effect on the actions of contraction-induced oxidants for a considerable time, although evidence for any specific role in this area is lacking. A review of studies in this area suggests that vitamin E supplements are unlikely to reliably reduce the severity of contraction-induced muscle damage but, in contrast, appear capable of modulating redox-regulated adaptive responses to contractions. Full evaluation of the roles of oxidants and antioxidants such as vitamin E in responses of muscle to contractions should enable the manipulation of these processes with potential beneficial effects on maintenance of optimal muscle function.

Effect of vitamin C supplements on antioxidant defence and stress proteins in human lymphocytes and skeletal muscle.

Oxidative stress induces adaptations in the expression of protective enzymes and heat shock proteins (HSPs) in a variety of tissues. We have examined the possibility that supplementation of subjects with the nutritional antioxidant, vitamin C, influences the ability of lymphocytes to express protective enzymes and HSPs following exposure to an exogenous oxidant and the response of skeletal muscle to the physiological oxidative stress that occurs during exercise in vivo. Our hypothesis was that an elevation of tissue vitamin C content would reduce oxidant-induced expression of protective enzymes and HSP content. Lymphocytes from non-supplemented subjects responded to hydrogen peroxide with increased activity of superoxide dismutase (SOD) and catalase, and HSP60 and HSP70 content over 48 h. Vitamin C supplementation at a dose of 500 mg day-1 for 8 weeks was found to increase the serum vitamin C concentration by ~50 %. Lymphocytes from vitamin C-supplemented subjects had increased baseline SOD and catalase activities and an elevated HSP60 content. The SOD and catalase activities and the HSP60 and HSP70 content of lymphocytes from supplemented subjects did not increase significantly in response to hydrogen peroxide. In non-supplemented subjects, a single period of cycle ergometry was found to significantly increase the HSP70 content of the vastus lateralis. Following vitamin C supplementation, the HSP70 content of the muscle was increased at baseline with no further increase following exercise. We conclude that, in vitamin C-supplemented subjects, adaptive responses to oxidants are attenuated, but that this may reflect an increased baseline expression of potential protective systems against oxidative stress (SOD, catalase and HSPs).

Skeletal muscles of aged male mice fail to adapt following contractile activity.

Skeletal muscle adapts rapidly following exercise by the increased production of heat-shock proteins (HSPs). The aim of this study was to examine the ability of muscle from adult and aged mice to produce HSPs following non-damaging exercise. Adult and aged B6XSJL mice were anaesthetized and their hind limbs were subjected to isometric contractions. At different time points, muscles were analysed for HSP production by Western and Northern blotting and by electrophoretic mobility-shift assay. HSP protein and mRNA levels in muscles from adult mice increased significantly following exercise. This was not evident in muscles of aged mice. In contrast, binding of the transcription factor heat-shock factor 1 (HSF1) was not grossly altered in muscles of aged mice compared with adult mice. The data suggest that the inability of muscles of aged mice to produce HSPs appears to be due to alterations during gene transcription.

Damage to developing mouse skeletal muscle myotubes in culture: protective effect of heat shock proteins.

Damage to skeletal muscle occurs following excessive exercise, upon reperfusion following ischaemia and in disease states, such as muscular dystrophy. Key mechanisms by which damage is thought to occur include a loss of intracellular calcium homeostasis, loss of energy supply to the cell, increased activity of oxidising free radical-mediated reactions and activation of apoptosis pathways. An increased cellular content of heat shock proteins (HSPs) has been shown to protect skeletal muscle against some forms of damage, although the mechanistic basis of this protection is not clearly understood. The aim of this study was to establish a cell culture-based model of damage to C2C12 skeletal muscle cells using the calcium ionophore, A23187 and the mitochondrial uncoupler, 2,4-dinitrophenol (DNP) as damaging agents. Treatment of cells with 1 mM DNP for 60 min resulted in the release of 63.5 % of intracellular creatine kinase (CK) activity over the 3 h experimental period. Treatment of cells with 10 microM A23187 for 30 min resulted in the release of 47.9 % of CK activity. Exposure of myotubes to a period of hyperthermia resulted in a significant increase in their content of HSP25, HSP60, HSC70 (heat shock cognate) and HSP70. This increase in HSPs was associated with significant protection against both DNP-induced and A23187-induced damage to the myotubes. These results indicate that an increased content of HSPs may provide protection against the muscle damage that occurs by a pathological increase in intracellular calcium or uncoupling of the mitochondrial respiratory chain.

Somatostatin mediates nitric oxide production by activating sst(2) receptors in the rat retina.

Somatostatin and its receptors (ssts) are found in the retina. Recent evidence suggested the involvement of sst(2A) and sst(2B) receptors in the regulation of nitric oxide (NO) (). In this study, we investigated further the localization of sst(1), sst(3)-sst(5), and the possible involvement of all subtypes, present in the rat retina, in the regulation of NO production. Polyclonal antibodies raised against sst(1), sst(3-5) were applied to 10-14 micro m cryostat sections of rat retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. The levels of NO in rat retinal explants were assessed by the production of its stable metabolites NO(2)(-) and NO(3)(-). sst(1) immunofluorescence was detected mainly in the retinal pigment epithelium, blood vessels of the inner retina, where it was colocalized with NADPH-diaphorase, and in processes of the inner plexiform layer (IPL). sst(4) immunohistochemistry was found in ganglion cell bodies, where it was colocalized with NADPH-diaphorase, processes of the IPL and ganglion cell layer, and optic nerve fibers. sst(3) or sst(5) immunostain was not detected. Somatostatin increased NO production and this effect was mimicked only by the sst(2) specific analog L-779976. The sst(2) antagonist CYN-154806 blocked the L-779976 increase of NO production. These results present conclusive evidence that somatostatin's role in the retina involves the regulation of NO by an sst(2) mechanism.

NADPH-diaphorase colocalization with somatostatin receptor subtypes sst2A and sst2B in the retina.

PURPOSE: To investigate the differential localization of somatotropin release-inhibitory factor (SRIF) receptor subtypes (sst2A and sst2B) and their possible colocalization with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase in the rat and rabbit retina. METHODS: Polyclonal antibodies raised against sst2A and sst2B receptors were applied to 10- to 14-microm cryostat sections of rat and rabbit retinas fixed in paraformaldehyde. NADPH-diaphorase reactivity was assessed histochemically. Double labeling was performed for sst2A or sst2B receptors with NADPH-diaphorase, and with markers for the cell types present in the retina (protein kinase C [PKC], tyrosine hydroxylase; [TH], calbindin, and recoverin). RESULTS: sst2A immunoreactivity was detected in rod bipolar cells and colocalized with NADPH-diaphorase in the rabbit, but not the rat, retina. sst2B was present only in photoreceptor cells of the rat and colocalized with NADPH-diaphorase. CONCLUSIONS: These results suggest that SRIF, acting through sst2A receptors in bipolar cells and sst2B receptors in photoreceptor cells, may affect nitric oxide function in the rabbit and rat retina.