Do post-concussion-like symptom responses change following exercise or sports participation in a non-concussed cohort?

The purposes of this study were (a) to determine the reliable change in post-concussion-like symptoms reported following self-selected exercise or sports activities and (b) to explore the potential influence of gender and exercise parameters on post-concussion-like symptoms reported by a non-concussed cohort following exercise/training. A pre-to-post observational design was used. A convenience sample of students aged 18-30 years who visited a university recreation center to engage in their chosen exercise activity and a purposeful sample of men's and women's rugby union players engaged in their regular training sessions were included in the study. All participants reported their symptoms using the symptom scale of the Sport Concussion Assessment Tool 2. The reliable change index was used to determine the change in symptom scores reported from pre-to post-exercise/training. Multiple linear regression analysis was used to model the exercise variables to explain the impact on the reporting of symptoms. A total of 260 participants (146 males and 114 females) completed their self-selected exercise activity or rugby union training. Approximately two-thirds of all participants did not demonstrate a change (increase or decrease) in total symptom score (201/260, 77.9%) and/or symptom severity score (212/260, 81.9%) from pre-to post-exercise/training. The symptom response following exercise or sports training did not change in the majority of participants. Clinicians need to be aware of these findings to make informed decisions on return-to-play following a concussive brain injury.

Sports concussion assessment: the effect of exercise on dynamic and static balance.

This study determined the effect of exercise on measures of static and dynamic balance used in the assessment of sports-related concussion (SRC). A balanced three-group cross-over randomized design was used with three levels of exercise verified by blood-lactate, heart rate and "perceived-exertion": no exercise/rest (NE), moderate-intensity exercise (ME), and high-intensity exercise (HE). Participants performed two timed balance tasks: tandem gait (TG) and single-leg stance (SLS); pre- and post-exercise and 15 min after exercise. Linear mixed-models with adjusted means and contrasts compared exercise effects. Ninety asymptomatic participants (45♂:45♀) were recruited. When times were contrasted with NE; HE resulted in a significant decrease in SLS (P<0.001) and TG (P<0.001) performance immediately following exercise. Fifteen minutes of recovery improved SLS (P<0.001) and TG (P=0.011) from post-exercise performance. ME caused a significant decrease in performance in SLS (P=0.038) but not TG (P=0.428). No statistically significant change occurred following ME in any tasks after 15-min recovery (SLS P=0.064; TG P=0.495). Test-retest reliability was considerably higher for the dynamic task compared with the static task. The reliability of static and dynamic balance tasks, and the change in performance following exercise, have implications for the immediate assessment of SRC, as these measures are utilized in concussion assessment instruments.

Does the purchase of a mobility scooter alter physical activity levels in older adults?

Purpose: This cohort study investigated changes in physical activity, community mobility and social participation following the first-time purchase of a mobility scooter.Methods: A national sample of persons aged 65+ years was surveyed using customized semi-structured interviews that explored changes in physical activity via the International Physical Activity Questionnaire modified for the elderly, and community involvement. Participants were recruited at the point of purchase of their first mobility scooter, and interviewed at this time and again at 2 and 6 months post-purchase.Results: Eighteen participants (F = 10, M = 8) aged between 65 and 95 years were recruited. Physical activity levels remained unchanged in 12 participants, and declined by at least one category in five participants. All participants reported improvement to their self-perceived quality of life following acquisition of a mobility scooter. Participants did not access additional forms of physical activity, though nine reported increased social participation. At baseline, five participants stated that the scooter was used for journeys they formerly made by other motorized transport, and by the 6-month interview, this number had risen to 15 participants.Conclusions: It is unlikely that changes in physical activity were related to the ageing process given the relatively short time span of the study. Thus it can be inferred that participants viewed their mobility scooter as a vehicle for maintaining their lifestyle rather than as a means to seek out additional activities. Improvements to perceived quality of life may be attributed to continuing or furthering community and social engagement, and a sense of retained independence.Implications for rehabilitationAn awareness of possible changes in physical activity associated with the purchase of a mobility scooter is needed.The purchase of a mobility scooter provides a viable means of transport to facilitate access to physical activity situations.Over-reliance on a mobility scooter has the potential to decrease health-related physical activity.The development and dissemination of a targeted health message about maintaining (or improving) physical activity levels is warranted to increase the awareness in this growing group of new, and current, mobility scooter riders in an increasingly ageing population.

Chemotactic peptide receptor modulation in polymorphonuclear leukocytes.

The binding of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) to its receptor on rabbit polymorphonuclear leukocytes (PMNs) modulates the number of available peptide receptors. Incubation with FNLLP decreases subsequent binding capacity, a phenomenon that has been termed receptor down regulation. Down regulation of the chemotactic peptide receptor is concentration dependent in both the rate and extent of receptor loss. The dose response parallels that of FNLLP binding to the recptor. The time-course is rapid; even at concentrations of FNLLP as low as 3 x 10(-9) M, the new equilibrium concentration of receptors is reached within 15 min. Down regulation is temperature dependent, but does occur even at 4 degrees C. Concomitant with down regulation, some of the peptide becomes irreversibly cell associated. At 4 degrees C, there is a small accumulation of nondissociable peptide that rapidly reaches a plateau. At higher temperatures, accumulation of nondissociable peptide continues after the rceptor number has reached equilibrium, and the amount accumulated can exceed the initial number of receptors by as much as 300%. The dose response of peptide uptake at 37 degrees C reflects that of binding, suggesting that it is receptor mediated. This uptake may occur via a pinocytosis mechanism. Although PMNs have not been considered to be pinocytic, the addition of FNLLP causes a fourfold stimulation of the rate of pinocytosis as measured by the uptake of [3H]sucrose.

Sensory adaptation of leukocytes to chemotactic peptides.

The morphology and behavior of polymorphonuclear leukocytes (PMNs) were studied after rapid changes in the concentration of a chemotactic factor N-formylnorleucylleucylphenylalanine (f-NorleuLeuPhe) (Schiffmann et al., 1975, Proc. Natl. Acad. Sci. U. S. A. 72:1059--1062). After an increase in peptide concentration, the cells round, form lamellipodia or ruffles over most of their surface, and stop locomotion. These changes are transient. After a delay, the cells, still in the presence of peptide, withdraw most of the ruffles and resume locomotion, forming ruffles only at their front. Cells repeat the transient generalized ruffling upon further increase in peptide concentration. The behavioral changes occur over the same dose range as binding to a saturable receptor. The duration of the transient response after a concentration increase is roughly proportional to the increase in the number of cell receptors occupied as a result of the concentration change. Decreasing the concentration of peptide causes the cells to round transiently and form blebs before they recommence locomotion. The transient nature of these aspects of the cell's responsiveness to chemotactic factors appears to be due to adaptation by the cells. The ability to adapt to the concentration of a chemotactic factor may be important in leukocyte chemotaxis.

Asymmetric distribution of the chemotactic peptide receptor on polymorphonuclear leukocytes.

The distribution of chemotactic peptide receptors on polymorphonuclear leukocytes (PMNs) was visualized using tritiated chemotactic peptide, N-formylmethionyl-leucylphenylalanine, coupled to hemocyanin (HY-FMLP). This probe was biologically active and the number of HY-FMLP molecules bound to the cell in a saturable manner corresponded closely to the number of peptide receptors characterized for rabbit peritoneal polymorphonuclear leukocytes (Sullivan, S. J., and S. H. Zigmond, 1980, J. Cell Biol., 85:703-711). Cells exhibiting locomotion have a polar morphology easily recognized in the scanning electron microscope. HY-FMLP bound to these cells was asymmetrically distributed with the highest density of HY-FMLP bound to the midregion of the cell. There were very few particles bound to the tail regions. The binding to the leading ruffles was variable but usually less than to the midregion. Addition of high concentrations of uncoupled FMLP eliminated HY-FMLP binding, confirming that the hemocyanin observed was a marker for the saturable chemotactic peptide receptor. The asymmetry in receptor distribution was seen on cells that had been stimulated by low concentrations of either FMLP or another chemotactic factor, leukotriene B4. Thus, peptide binding to the receptor was not required for the development of the asymmetric distribution. The low density of receptors in the tail region of the cell was consistent with the decreased responsiveness of the tail to chemotactic stimulation (Zigmond, S. H., H. I. Levitsky, and B. J. Kreel, 1981, J. Cell Biol., 89:585-592). The receptor asymmetry may contribute to the polar behavior exhibited by polymorphonuclear leukocytes and would be expected to quantitatively modify the directional information available to a cell in a gradient of chemotactic peptide.

Kinetic analysis of chemotactic peptide receptor modulation.

The dynamics of the chemotactic peptide receptor on rabbit peritoneal polymorphonuclear leucocytes were followed using the tritiated peptide N-formylnorleucylleucylphenylalanine (FNLLP). We have used a kinetic analysis to examine the possible interrelationships between receptor loss (down-regulation), receptor-mediated peptide uptake, and receptor recycling. We have previously demonstrated that cells incubated with FNLLP show a dose-dependent reduction in the number of receptors available on the surface. This receptor down-regulation is complete within 20 min and then the number of receptors available for binding remains at a plateau level. Peptide continues to be taken up in a receptor-mediated manner even after down-regulation is complete. If peptide is removed, receptor recovery occurs and does not require protein synthesis. In these studies we have investigated the kinetics of these processes. On the basis of this analysis, we propose that the plateau receptor level is a steady-state in which receptor internalization and return occur continuously. We demonstrate that the rate of receptor-mediated peptide uptake is approximately equal to the rate of receptor recovery measured after peptide removal. In addition, the rate of receptor recovery is proportional to the number of receptors missing from the surface, suggesting receptor recycling may be occurring.

Biohybrid artificial pancreas: long-term implantation studies in diabetic, pancreatectomized dogs.

Diabetic complications such as neuropathy, retinopathy, and renal and cardiovascular disease continue to pose major health risks for diabetic patients. Consequently, much effort has focused on approaches that could replace conventional insulin therapy and provide more precise regulation of blood glucose levels. The biohybrid perfused artificial pancreas was designed to incorporate islet tissue and a selectively permeable membrane that isolates this tissue from the immune system of the recipient. Biohybrid pancreas devices containing canine islet allografts were implanted in ten pancreatectomized dogs requiring 18 to 32 units of injected insulin daily. These implants resulted in good control of fasting glucose levels in six of these animals without further exogenous insulin for periods of up to 5 months.

Self-report scales/checklists for the measurement of concussion symptoms: a systematic review.

OBJECTIVE: To identify self-reported sport concussion symptom scales and to describe the psychometric properties of these identified scales. DESIGN: Systematic review. INTERVENTION: PubMed, Medline, CINAHL, Scopus, Web of Science, Sport Discus, PsycINFO and AMED were searched from their establishment until December 2008. The medical subject heading terms "brain concussion", "signs or symptoms" and "athletic injuries". The search was limited to articles published in English. An additional search of the reference lists of the retrieved articles was conducted. Only full-text articles were considered for this study and these were retrieved to determine whether they met the inclusion criteria. RESULTS: The initial search resulted in 421 articles, which were reduced to 290 articles after removing duplicates. The hand search resulted in 17 articles, thus giving a total of 307 articles. Full text was available for 295 articles of which 60 met the criteria for inclusion. The excluded 235 articles were case reports, reviews and guidelines on concussion management or studies that had not used a symptom scale or checklist. CONCLUSIONS: Six core scales were identified with a broad range of symptom items but with limited information on their psychometric properties. There were numerous derivative scales reported, most of which have not been methodically developed or subjected to scientific scrutiny. Despite this, they do make a contribution to the detection, assessment and return to play decisions but there is a need for the clinical user to be aware that many of these scales have "evolved" rather than being scientifically developed.

Physiotherapists' use of information in identifying a concussion: an extended Delphi approach.

OBJECTIVE: To determine the relative importance of signs and symptoms that a selected cohort of sports physiotherapists use to identify a sports concussion. METHODS: A two-round Delphi methodology was used to achieve consensus in a cohort of 21 sports physiotherapists. A subsequent round involving an educational intervention provided the participants with an opportunity to modify their knowledge base through the provision of a relevant resource article. RESULTS: Participants provided 123 responses, which were grouped into eight descriptive categories with consensus (>80%) being reached for the importance of: cognition/orientation, memory, motor dysfunction and state of consciousness. The category "state of consciousness" remained the most important information source at the completion of the study. CONCLUSION: Participants placed considerable importance on the player's level of consciousness in their decision making. This would appear to be in conflict with recent trends to place greater importance on the role of symptoms in identifying a concussion.

The effect of exercise on motor performance tasks used in the neurological assessment of sports-related concussion.

Sports-related concussion is assessed using both cognitive and motor performance tasks. There is limited understanding of how exercise affects these measures. The purpose of this study was to investigate the effect of moderate-intensity exercise on three selected measures of motor performance. A repeated measures design was used to compare baseline motor performance scores with post-exercise scores with an exercise intervention modelled on the physiological demands of a team sport. 30 physically active subjects performed timed motor performance tasks: Finger-to-Nose (FTN), Tandem Gait (TG) and Single Leg Stance (SLS). The tasks were administered twice pre-exercise and twice post-exercise. FTN, TG and SLS demonstrated high test-retest reliability (ICC values >0.8). 15 minutes of moderate-intensity exercise caused a significant improvement in FTN (T2 = 2.66 (SD 0.38), T3 = 2.49 (0.32); p<0.001) and TG (T2 = 13.08 (2.84), T3 = 12.23 (2.22); p = 0.001), but not in SLS (T2 = 5.94 (4.99), T3 = 5.91 (5.54); p = 0.507). Improvement in the performance of motor tasks after exercise has implications for the immediate assessment of sports-related concussion, given that measures of motor performance are utilised in concussion assessment instruments.

A biopsychosocial understanding of lower back pain: Content analysis of online information.

OBJECTIVES: (1) To develop a checklist to assess the representation of biopsychosocial lower back pain (LBP) online information; (2) to analyse publicly accessed online LBP information from a Google search for the degree that psychosocial contributors are described alongside the traditional biomedical approach to explaining LBP; (3) whether websites use information on pain biology to educate on LBP; (4) any inaccurate or false information regarding the mechanisms of LBP and; (5) the amount of websites certified by established benchmarks for quality health information. MATERIALS AND METHODS: An online search was conducted using the Google search engines of six major English-speaking countries. Website content was analysed using three checklists developed for the purpose of this study - Biopsychosocial information categorisation checklist and scoring criteria; pain biology information checklist; and the inaccurate information checklist. Website quality was identified by the presence of an Health on the Net certification (HONcode). RESULTS: Of the fifteen websites analysed, the content of 26.7% of websites was classified as 'biomedical', 60% 'limited psychosocial' and 13.3% 'reasonable psychosocial'; 20% included information on pain biology; 46.7% inaccurately implied pain to be equal to tissue damage and 46.7% implied pathways specific to pain transmission; 40% were HONcode certified. CONCLUSION: Online LBP information retrieved through a Google search has limited to no integration of psychosocial or pain biology information. The focus on tissue pathology is further supported by the inaccurate descriptions of pain as equal to tissue damage and as an input to the central nervous system (CNS). Online LBP information needs to be guided by criteria more sensitive to the psychosocial contributors to pain. SIGNIFICANCE: The online LBP information retrieved from a Google search needs to be guided by information more sensitive to the psychosocial contributors to pain and disability. This study also highlights the presence of inaccurate information that implied pain as a measure of tissue damage or as an input to the nervous system.

Toddlers' Differential Susceptibility to the Effects of Coparenting on Social-Emotional Adjustment.

The present study tested whether infants high in negative affectivity are differentially susceptible to observed coparenting behavior in relation to their subsequent social-emotional development. Data came from a longitudinal study of 182 U.S. dual-earner, primiparous couples and their infant children. At 9-months postpartum, child negative affectivity was reported by mothers and fathers and supportive and undermining coparenting behavior were assessed from mother-father-infant observations. At 27-months mothers reported on toddlers' externalizing behavior and dysregulation using a clinical assessment tool designed to identify competencies and areas of concern in toddlers' social-emotional development. Hierarchical regression analyses revealed partial support for the differential susceptibility hypothesis. Specifically, infants high in negative affectivity had lower levels of dysregulation when embedded in a more supportive coparenting context, and higher levels of dysregulation when embedded in a less supportive coparenting context. In contrast, supportive coparenting behavior was not relevant for the dysregulation of infants initially low in negative affectivity.

Uncoupling of M-phase kinase activation from the completion of S-phase by heat shock.

Chronic exposure of asynchronous HeLa cell cultures to 41.5 degrees C leads to an accumulation of cells in the S-phase, spontaneous premature chromosome condensation, and loss of clonogenicity (M.A. Mackey, S. L. Anolik, and J. L. Roti Roti. Cancer Res., 52: 1101-1106, 1992). In this report, we show that increases in histone H1 kinase activity during 41.5 degrees C exposure occur coincidentally with the appearance of premature chromosome condensation. Furthermore, this kinase activity is shown to be associated with M-phase kinase complexes containing cyclin B1. These increases in the activity of M-phase kinase were found to occur concomitantly with an elevation in cyclin B1 mRNA and an accumulation of cyclin B1 protein. Because cyclin B1 transcription begins in the S-phase, it is probable that the heat-induced delay in the S-phase allows the accumulation of abnormally high cyclin B1 levels. Elevated cyclin B1 levels could then account for the observed abrogation of the cell cycle checkpoint, which usually assures that mitosis does not proceed until DNA replication is complete. This involvement of M-phase kinase in heat-induced cytotoxicity demonstrates the importance of the coordinate regulation of the processes of DNA replication and entry into mitosis.

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress.

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.

Perspectives in diabetes. Islet transplantation with immunoisolation.

Immunoisolation is a potentially important approach to transplanting islets without need for immunosuppressive drugs. Immunoisolation systems have been conceived in which the transplanted tissue is separated from the immune system of the host by an artificial barrier. These systems offer a solution to the problem of human islet procurement by permitting use of islets isolated from animal pancreases. The devices used are referred to as biohybrid artificial organs because they combine synthetic, selectively permeable membranes that block immune rejection with living transplants. Three major types of biohybrid pancreas devices have been studied. These include devices anastomosed to the vascular system as AV shunts, diffusion chambers, and microcapsules. Results in diabetic rodents and dogs indicate that biohybrid pancreas devices significantly improve glucose homeostasis and can function for more than a year. Recent progress made with this approach is discussed, and some of the remaining problems that must be resolved to bring this technology to clinical reality are addressed.

Treatment of severely diabetic pancreatectomized dogs using a diffusion-based hybrid pancreas.

Long-term survival of dog islet allografts implanted in diabetic pancreatectomized dogs was achieved by islet encapsulation inside cylindrical chambers fabricated from permselective acrylic membranes (nominal M(r) exclusion of 50,000-80,000). Dog islets were isolated from the pancreases of outbred mongrel dogs by collagenase digestion. Chambers containing mean +/- SE 316 +/- 63K islet equivalents (mean islet volume, 558 +/- 111 mm3, purity 90-95%) were peritoneally implanted into six totally pancreatectomized dogs. The dogs were monitored for glycemic control by fasting and postprandial blood glucose determinations, and responses to both intravenous glucose (intravenous glucose tolerance test 0.5 g/kg) and oral glucose (oral glucose tolerance test 1 g/kg). All of the dogs required appreciably lower dosages of exogenous insulin therapy for control of fasting blood glucose levels, with the mean daily insulin dose dropping from 38 +/- 7 to 5 +/- 1 U/day during the 1st wk. Three recipients required no insulin for greater than 82, greater than 68, and 51 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 and 2 mo postimplantation were 2.7 +/- 0.4 and 2.0 +/- 0.5, respectively compared with 3.5 +/- 0.5 before pancreatectomy. The glucose values during oral glucose tolerance tests at 2 wk, although returning to less than 125 mg/dl (less than 7.0 mM) by 2 h, exceeded the normal range, with peak values of 174 to 202 mg/dl (9.7 to 11.3 mM). These preliminary results are encouraging, and represent an important step in determining the feasibility of using this type of diffusion-based hybrid artificial pancreas as treatment for diabetes mellitus in humans.

Distribution of somatostatin receptors in RINm5F insulinoma cells.

Previous studies with heterogeneous populations of pancreatic cells have provided evidence for the presence of somatostatin (SRIF) receptors in cytosol and secretion vesicles, as well as the plasma membrane. To examine the distribution of SRIF receptors between soluble and membrane fractions in a homogeneous pancreatic islet cell population, we have used the clonal RINm5F insulinoma cell line. These cells contain specific, high affinity binding sites for [125I-Try11]SRIF on the cell surface, and occupancy of these sites by SRIF and SRIF analogs correlates with inhibition of insulin secretion. Stable, steady state binding was achieved using both intact cells and membranes by performing binding incubations with [25I-Tyr11]SRIF at 22 C. Half-maximal inhibition of [125I-Tyr11]SRIF binding occurred with 0.21 +/- 0.11 nM SRIF in membranes and 0.35 +/- 0.30 nM SRIF in cells. In contrast, the binding of [125I-Tyr11]SRIF to cytosolic macromolecules was not reduced by concentrations of SRIF as high as 100 nM, demonstrating that this binding was of much lower affinity. RINm5F membranes were further purified using a Percoll gradient to prepare a microsomal fraction, which was enriched in adenylate cyclase activity, and a secretory granule fraction, which was enriched in insulin. [125I-Tyr11]SRIF binding to the microsomal fraction (3.8 +/- 0.3 fmol/mg) was 3 times higher than to secretion granules (1.2 +/- 0.2 fmol/mg). Thus, high affinity SRIF binding sites were most abundant in microsomal membranes and were low or undetectable in secretory granules and cytosol. To determine whether translocation of SRIF receptors to the plasma membrane accompanied insulin secretion, we examined the effects of various insulin secretagogues on [125I-Tyr11]SRIF binding to intact cells. Leucine (20 mM), glyceraldehyde (15 mM), forskolin (1 microM), and glucagon (1 microM) stimulated insulin release 1.5- to 4.0-fold in different experiments. However, these secretagogues did not increase [125I-Tyr11]SRIF binding. In summary, our results indicate that high affinity SRIF receptors in RINm5F cells are located primarily on the plasma membrane and that the concentration of SRIF receptors at the cell surface is independent of the secretory activity of the cells.

Characterization of somatostatin receptors which mediate inhibition of insulin secretion in RINm5F insulinoma cells.

Somatostatin (SRIF) is a neuropeptide which inhibits secretion from a variety of target cells including pancreatic beta-cells. In this study we have used the RINm5F rat insulinoma cell line to characterize high affinity receptors for SRIF. The binding of 0.03 nM [125I-Tyr11]SRIF to RINm5F cells reached a plateau level within 4 h at 37 C at which time 80% of the total binding could be displaced by 100 nM unlabeled SRIF. In contrast, 100 nM concentrations of eight structurally unrelated peptides did not inhibit [125I-Tyr11]SRIF binding. Scatchard analysis indicated that RINm5F cells contained a single class of noninteracting binding sites (910 +/- 190 sites per cell) with high affinity for [125I-Tyr11]SRIF [equilibrium dissociation constant (Kd) = 0.04 +/- 0.01 nM]. Competition experiments with SRIF analogs showed that the binding affinity for [I-Tyr11]SRIF (Kd = 0.03 +/- 0.02 nM) was higher than that for either SRIF (0.24 +/- 0.04 nM) or [Tyr11]SRIF (0.27 +/- 0.04 nM) and that reduced SRIF analogs bound poorly (Kd greater than 50 nM). These results demonstrate that RINm5F cells possess specific, high affinity binding sites for SRIF. Insulin release stimulated by 20 mM leucine or 15 mM glyceraldehyde was inhibited as much as 80% by maximal concentrations (100 nM) of SRIF. The IC50 for SRIF inhibition of leucine-stimulated insulin secretion was 0.43 +/- 0.15 nM, in good agreement with the apparent Kd for binding. In fact, this close correlation between binding affinity and potency to inhibit insulin release was observed for six SRIF analogs, indicating that the characterized binding sites are the receptors which mediate the biological actions of SRIF in RINm5F cells.

Glutathione dependent metabolism and detoxification of 4-hydroxy-2-nonenal.

The involvement of glutathione (GSH) dependent processes in the detoxification of 4-hydroxy-2-nonenal (4HNE) was investigated using Chinese hamster fibroblasts and clonogenic cell survival. GSH reacted, in a dose-dependent fashion, with 4HNE in phosphate buffer at pH 6.5, leading to the disappearance of 4HNE. The addition of glutathione transferase activity (GST) facilitated a more rapid disappearance of 4HNE but the reaction was still dependent on the concentration of GSH. When cell cultures were exposed to the reaction mixtures, 4HNE cytotoxicity was also reduced in a manner which was dependent on the concentration of GSH. When 2.16- or 1.08-mM GSH were incubated in phosphate buffer with 1.08-mM 4HNE in the presence or absence of GST, then mixed with media and placed on cells for 1 h, the cytotoxicity associated with exogenous exposure to free 4HNE was abolished. GSH depletion (greater than 90%) using buthionine sulfoximine (BSO) was accomplished in control (HA1) and H2O2-resistant variants derived from HA1. GSH depletion resulted in enhanced cytotoxicity of 4HNE in all cell lines. This BSO-induced sensitization to 4HNE cytotoxicity was accompanied by a significant reduction in the ability of cells to metabolize 4HNE. The magnitude of the sensitization to 4HNE toxicity caused by GSH depletion was similar to the magnitude of the reduction in the ability of cells to metabolize 4HNE. These results support the hypothesis that GSH and GST provide a biologically significant pathway for protection against aldehydic by-products of lipid peroxidation.