Factors affecting sports involvement in a school-based youth cohort: Implications for long-term athletic development.

The aim of the present study was to determine the factors affecting sports involvement in a school-based adolescent population. The cross-sectional cohort study assessed anthropometry, physical capacities and motor competence in 501 boys (aged 10-16 y), from junior (10-12 y) and senior (13-16 y) cohorts. Sports participation data was collected from junior participants. Multivariate analysis of covariance revealed moderate maturity, anthropometry, physical capacity and motor competence differences between sports in the senior cohort (F = 2.616, p < 0.001, η2 = .08), but not in the junior cohort. Furthermore, differences in physical fitness were revealed between playing levels (F = 2.616, p < 0.001, η2 = .08), with a discriminant analysis correctly classifying 73% of participants using aerobic fitness and vertical jump measures. Representative level participants engaged in more structured training and commenced organised competition at a later age (F = 4.332, p < 0.001, η2 = .21). This study's findings are twofold: 1) physical and motor competence profiles differ more between sports with increasing age, and 2) participants at a higher level of competition report delayed engagement in their main sport. As a result, schools may be the ideal environment in which to provide children and adolescents with the opportunity to sample different sports.

Heat-acclimatization and pre-cooling: a further boost for endurance performance?

To determine if pre-cooling (PC) following heat-acclimatization (HA) can further improve self-paced endurance performance in the heat, 13 male triathletes performed two 20-km cycling time-trials (TT) at 35 °C, 50% relative humidity, before and after an 8-day training camp, each time with (PC) or without (control) ice vest PC. Pacing strategies, physiological and perceptual responses were assessed during each TT. PC and HA induced moderate (+10 ± 18 W; effect size [ES] 4.4 ± 4.6%) and very large (+28 ± 19 W; ES 11.7 ± 4.1%) increases in power output (PO), respectively. The overall PC effect became unclear after HA (+4 ± 14 W; ES 1.4 ± 3.0%). However, pacing analysis revealed that PC remained transiently beneficial post-HA, i.e., during the first half of the TT. Both HA and PC pre-HA were characterized by an enhanced PO without increased cardio-thermoregulatory or perceptual disturbances, while post-HA PC only improved thermal comfort. PC improved 20-km TT performance in unacclimatized athletes, but an 8-day HA period attenuated the magnitude of this effect. The respective converging physiological responses to HA and PC may explain the blunting of PC effectiveness. However, perceptual benefits from PC can still account for the small alterations to pacing noted post-HA.

Elective ICP monitoring: how long is long enough?

BACKGROUND: Intracranial pressure monitoring is commonly undertaken to assess and manage acute patients following head injury. However, ICP monitoring can also be a useful diagnostic tool in the management of CSF dynamics in elective patients. To date, there is little published research to suggest how long these elective patients require ICP monitoring in order to gain an accurate picture of a patient's ICP dynamics. At the author's institution, a minimum of 48-h data collection is currently undertaken in patients with a suspected ICP abnormality. METHODS: A retrospective audit was undertaken comparing overall median ICP and overall median pulse amplitude data at three time points, 24 h, 48 h and total time analysed (if longer than 48 h). Paired T-test was used to assess if there were statistically significant differences between 24-h versus 48-h monitoring and total duration of monitoring. All patients admitted over a 6-month period for ICPM who met the inclusion/exclusion criteria were included. RESULTS: Eighteen patients met the criteria. Median age was 45.8 years, range 22-83 years, 12 female and 6 male. No complications were experienced as a result of ICPM. Diagnosis included NPH, IIH, suspected shunt malfunction and Chiari malformation. The results demonstrated that there is no statistical difference between 24 h and 48 h or longer for both overall median ICP and pulse amplitude. CONCLUSION: The results of this study demonstrate that ICP monitoring of elective adult patients using a Spiegelberg intraparenchymal bolt for 24 h gives an accurate picture of a patient's ICP dynamics compared with longer periods of monitoring.

Monitoring training to assess changes in fitness and fatigue: The effects of training in heat and hypoxia.

This study examined the association between monitoring tools, training loads, and performance in concurrent heat and hypoxia (H + H) compared with temperate training environments. A randomized parallel matched-group design involved 18 well-trained male cyclists. Participants performed 12 interval sessions (3 weeks) in either H + H (32 ± 1 °C, 50% RH, 16.6% O2 normobaric hypoxia) or control (21 °C, 50% RH, 21% O2 ), followed by a seven-session taper (3 weeks; 21 °C, 50% RH, 21% O2 ), while also maintaining external training (∼ 6-10 h/week). A 20-km time trial (TT) was completed pre- and post-training block (21 °C, 50% RH, 21% O2 ). Before each TT and once weekly, a 4-min cycle warm-up (70% 4-min mean maximum power) was completed. Visual analog scale rating for pain, recovery, and fatigue was recorded before the warm-up, with heart rate (HREx ), heart rate recovery (HRR), and rating of perceived exertion (RPEWU ) recorded following. Training load was quantified using the session rating of perceived exertion (sRPE) method throughout. Overall TT improved 35 ± 47 s with moderate correlations to HRR (r = 0.49) and recovery (r = -0.55). H + H group had a likely greater reduction in HREx [ES = -0.50 (90% CL) (-0.88; 0.12)] throughout and a greater sRPE (ES = 1.20 [0.41; 1.99]), and reduction in HRR [ES = -0.37 (-0.70;-0.04)] through the overload. RPEWU was associated with weekly training load (r = 0.37). These findings suggest that recovery and HRR in a temperate environment may be used as simple measures to identify an athlete's readiness to perform. Alternatively, the relationship of RPEWU and training load suggests that perception of effort following a standardized warm-up may be a valid measure when monitoring an athlete's training response, irrespective of the training environment.

Consensus recommendations on training and competing in the heat.

Exercising in the heat induces thermoregulatory and other physiological strain that can lead to impairments in endurance exercise capacity. The purpose of this consensus statement is to provide up-to-date recommendations to optimize performance during sporting activities undertaken in hot ambient conditions. The most important intervention one can adopt to reduce physiological strain and optimize performance is to heat acclimatize. Heat acclimatization should comprise repeated exercise-heat exposures over 1-2 weeks. In addition, athletes should initiate competition and training in a euhydrated state and minimize dehydration during exercise. Following the development of commercial cooling systems (e.g., cooling vest), athletes can implement cooling strategies to facilitate heat loss or increase heat storage capacity before training or competing in the heat. Moreover, event organizers should plan for large shaded areas, along with cooling and rehydration facilities, and schedule events in accordance with minimizing the health risks of athletes, especially in mass participation events and during the first hot days of the year. Following the recent examples of the 2008 Olympics and the 2014 FIFA World Cup, sport governing bodies should consider allowing additional (or longer) recovery periods between and during events for hydration and body cooling opportunities when competitions are held in the heat.

Consensus recommendations on training and competing in the heat.

Exercising in the heat induces thermoregulatory and other physiological strain that can lead to impairments in endurance exercise capacity. The purpose of this consensus statement is to provide up-to-date recommendations to optimise performance during sporting activities undertaken in hot ambient conditions. The most important intervention one can adopt to reduce physiological strain and optimise performance is to heat acclimatise. Heat acclimatisation should comprise repeated exercise-heat exposures over 1-2 weeks. In addition, athletes should initiate competition and training in a euhydrated state and minimise dehydration during exercise. Following the development of commercial cooling systems (eg, cooling-vest), athletes can implement cooling strategies to facilitate heat loss or increase heat storage capacity before training or competing in the heat. Moreover, event organisers should plan for large shaded areas, along with cooling and rehydration facilities, and schedule events in accordance with minimising the health risks of athletes, especially in mass participation events and during the first hot days of the year. Following the recent examples of the 2008 Olympics and the 2014 FIFA World Cup, sport governing bodies should consider allowing additional (or longer) recovery periods between and during events, for hydration and body cooling opportunities, when competitions are held in the heat.

Accuracy of GPS devices for measuring high-intensity running in field-based team sports.

We compared the accuracy of 2 GPS systems with different sampling rates for the determination of distances covered at high-speed and metabolic power derived from a combination of running speed and acceleration. 8 participants performed 56 bouts of shuttle intermittent running wearing 2 portable GPS devices (SPI-Pro, GPS-5 Hz and MinimaxX, GPS-10 Hz). The GPS systems were compared with a radar system as a criterion measure. The variables investigated were: total distance (TD), high-speed distance (HSR>4.17 m·s(-1)), very high-speed distance (VHSR>5.56 m·s(-1)), mean power (Pmean), high metabolic power (HMP>20 W·kg(-1)) and very high metabolic power (VHMP>25 W·kg(-1)). GPS-5 Hz had low error for TD (2.8%) and Pmean (4.5%), while the errors for the other variables ranged from moderate to high (7.5-23.2%). GPS-10 Hz demonstrated a low error for TD (1.9%), HSR (4.7%), Pmean (2.4%) and HMP (4.5%), whereas the errors for VHSR (10.5%) and VHMP (6.2%) were moderate. In general, GPS accuracy increased with a higher sampling rate, but decreased with increasing speed of movement. Both systems could be used for calculating TD and Pmean, but they cannot be used interchangeably. Only GPS-10 Hz demonstrated a sufficient level of accuracy for quantifying distance covered at higher speeds or time spent at very high power.

Adding heat to the live-high train-low altitude model: a practical insight from professional football.

OBJECTIVES: To examine with a parallel group study design the performance and physiological responses to a 14-day off-season 'live high-train low in the heat' training camp in elite football players. METHODS: Seventeen professional Australian Rules Football players participated in outdoor football-specific skills (32 ± 1°C, 11.5 h) and indoor strength (23 ± 1°C, 9.3 h) sessions and slept (12 nights) and cycled indoors (4.3 h) in either normal air (NORM, n=8) or normobaric hypoxia (14 ± 1 h/day, FiO2 15.2-14.3%, corresponding to a simulated altitude of 2500-3000 m, hypoxic (HYP), n=9). They completed the Yo-Yo Intermittent Recovery level 2 (Yo-YoIR2) in temperate conditions (23 ± 1°C, normal air) precamp (Pre) and postcamp (Post). Plasma volume (PV) and haemoglobin mass (Hb(mass)) were measured at similar times and 4 weeks postcamp (4WPost). Sweat sodium concentration ((Na(+))(sweat)) was measured Pre and Post during a heat-response test (44°C). RESULTS: Both groups showed very large improvements in Yo-YoIR2 at Post (+44%; 90% CL 38, 50), with no between-group differences in the changes (-1%; -9, 9). Postcamp, large changes in PV (+5.6%; -1.8, 5.6) and (Na(+))sweat (-29%; -37, -19) were observed in both groups, while Hb(mass) only moderately increased in HYP (+2.6%; 0.5, 4.5). At 4WPost, there was a likely slightly greater increase in Hb(mass) (+4.6%; 0.0, 9.3) and PV (+6%; -5, 18, unclear) in HYP than in NORM. CONCLUSIONS: The combination of heat and hypoxic exposure during sleep/training might offer a promising 'conditioning cocktail' in team sports.

Relative age, biological maturation and anaerobic characteristics in elite youth soccer players.

Being relatively older and having an advanced biological maturation status have been associated with increased likelihood of selection in young elite soccer players. The aims of the study were to investigate the presence of a relative age effect (RAE) and the influence of birth quarter on anthropometry, biological maturity and anaerobic parameters in 374 elite Belgian youth soccer players. The sample was divided into 3 age groups, each subdivided into 4 birth quarters (BQ). Players had their APHV estimated and height, weight, SBJ, CMJ, sprint 5 and 30 m were assessed. Overall, more players were born in BQ1 (42.3%) compared with players born in BQ4 (13.7%). Further, MANCOVA revealed no differences in all parameters between the 4 BQ's, controlled for age and APHV. These results suggest that relatively youngest players can offset the RAE if they enter puberty earlier. Furthermore, the results demonstrated possible differences between BQ1 and BQ4, suggesting that caution is necessary when estimating differences between players because of large discrepancies between statistical and practical significance. These findings also show that coaches should develop realistic expectations of the physical abilities of younger players and these expectations should be made in the context of biological characteristics rather than chronological age-based standards.

Monitoring training loads, stress, immune-endocrine responses and performance in tennis players.

The study aim was to investigate the effect of a periodised pre-season training plan on internal training load and subsequent stress tolerance, immune-endocrine responses and physical performance in tennis players. Well-trained young tennis players (n = 10) were monitored across the pre-season period, which was divided into 4 weeks of progressive overloading training and a 1-week tapering period. Weekly measures of internal training load, training monotony and stress tolerance (sources and symptoms of stress) were taken, along with salivary testosterone, cortisol and immunoglobulin A. One repetition maximum strength, running endurance, jump height and agility were assessed before and after training. The periodised training plan led to significant weekly changes in training loads (i.e. increasing in weeks 3 and 4, decreasing in week 5) and post-training improvements in strength, endurance and agility (P < 0.05). Cortisol concentration and the symptoms of stress also increased in weeks 3 and/or 4, before returning to baseline in week 5 (P < 0.05). Conversely, the testosterone to cortisol ratio decreased in weeks 3 and 4, before returning to baseline in week 5 (P < 0.05). In conclusion, the training plan evoked adaptive changes in stress tolerance and hormonal responses, which may have mediated the improvements in physical performance.

Changes in perceived stress and recovery in overreached young elite soccer players.

The aim of this study was to prospectively monitor sport-specific performance and assess the stress-recovery balance in overreached (OR) soccer players and controls. During two competitive seasons, 94 players participated in the study. The stress-recovery balance (RESTQ-Sport) and sport-specific performance (Interval Shuttle Run Test) were assessed monthly. Seven players with performance decrement of at least a month were classified as OR. Stress and recovery measures were assessed between groups (OR vs healthy players) and at different times within the OR group. An unfavorable total recovery score appeared 2 months before diagnosis when compared with the reference values of the healthy group established at the start of the season (P=0.009) and also over the two seasons (P=0.028). The scales Emotional Stress (P=0.044), Physical Recovery (P=0.009), General Well-being (P=0.001) and Sleep Quality (P=0.045) were sensitive to OR compared with the average of the healthy group over the two seasons. Finally, Fatigue and Being in Shape demonstrated the largest changes in stress and recovery within the OR players (effect size=1.14 and 1.50). The longitudinal monitoring of performance and changes in stress and recovery may be useful for the detection of OR in its earliest stage. The information obtained from these tests can be used to optimize individual training and recovery programs.

Relative age effect and Yo-Yo IR1 in youth soccer.

The aims of the study were to investigate the presence of a relative age effect and the influence of birth quarter on anthropometric characteristics, an estimation of biological maturity and performance in the Yo-Yo Intermittent Recovery Test level 1 in 606 elite, Flemish youth soccer players. The sample was divided into 5 chronological age groups (U10-U19), each subdivided into 4 birth quarters. Players had their APHV estimated and height, weight and Yo-Yo IR1 performance were assessed. Differences between quarters were investigated using uni- and multivariate analyses. Overall, significantly (P<0.001) more players were born in the first quarter (37.6%) compared to the last (13.2%). Further, no significant differences in anthropometric variables and Yo-Yo IR1 performance were found between the 4 birth quarters. However, there was a trend for players born in the first quarter being taller and heavier than players born in the fourth quarter. Players born in the last quarter tended to experience their peak in growth earlier, this may have enabled them to compete physically with their relatively older peers. Our results indicated selection procedures which are focused on the formation of strong physical and physiological homogeneous groups. Relative age and individual biological maturation should be considered when selecting adolescent soccer players.

Nutritional practices of elite swimmers during an intensified training camp: with particular reference to antioxidants.

AIM: Athletes should match their energy intake with expenditure in order to maintain lean body mass. It is also important to consume adequate amounts of antioxidant vitamins and minerals to maintain health. METHODS: To assess the dietary habits of six nationally ranked Australian swimmers physical training load and dietary intake (24 h food recall) and were recorded on a daily basis during a 4 day intensive physical training period. RESULTS: The results showed no significant difference between energy intake and expenditure (P=0.58) or the amount of carbohydrate consumed (P=0.14) compared to the Australian recommended daily intake (RDI). Athletes reported a significantly greater intake of vitamin A (P<0.01), vitamin C (P<0.01), vitamin E (P<0.01) and protein (P<0.01) than the RDI. CONCLUSION: It was concluded that these elite swimmers have an adequate dietary intake to allow for optimal physical training and performance.

What Role Do Chronic Workloads Play in the Acute to Chronic Workload Ratio? Time to Dismiss ACWR and Its Underlying Theory.

AIM: The aim of this study was to examine the associations between the injury risk and the acute (AL) to chronic (CL) workload ratio (ACWR) by substituting the original CL with contrived values to assess the role of CL (i.e., the presence and implications of statistical artefacts). METHODS: Using previously published data, we generated a contrived ACWR by dividing the AL by fixed and randomly generated CLs, and we compared these results to real data. We also reproduced previously reported subgroup analyses, including dichotomising players' data above and below the median CL. Our analyses follow the same, previously published modelling approach. RESULTS: The analyses with original data showed effects compatible with higher injury risk for ACWR only (odd ratios, OR: 2.45, 95% CI 1.28-4.71). However, we observed similar effects by dividing AL by the "contrived" fixed and randomly generated CLs: OR 1.95 (1.18-3.52) dividing by 1510 (average CL); and OR ranging from 1.16 to 2.07, using random CL 1.53 (mean). Random ACWRs reduced the variance relative to the original AL and further inflated the ORs (mean OR 1.89, from 1.42 to 2.70). ACWR causes artificial reclassification of players compared to AL alone. Finally, neither ACWR nor AL alone confer a meaningful predictive advantage to an intercept-only model, even within the training sample (c-statistic 0.574/0.544 vs. 0.5 in both ACWR/AL and intercept-only models, respectively). DISCUSSION: ACWR is a rescaling of the explanatory variable (AL, numerator), in turn magnifying its effect estimates and decreasing its variance despite conferring no predictive advantage. Other ratio-related transformations (e.g., reducing the variance of the explanatory variable and unjustified reclassifications) further inflate the OR of AL alone with injury risk. These results also disprove the etiological theory behind this ratio and its components. We suggest ACWR be dismissed as a framework and model, and in line with this, injury frameworks, recommendations, and consensus be updated to reflect the lack of predictive value of and statistical artefacts inherent in ACWR models.

Hormonal, metabolic and perceptual responses to different resistance training systems.

AIM: The purpose of the present study was to compare the effect of different resistance training systems (Multiple-set [MS] and Pyramid [P]) on hormonal, metabolic and perceptual markers of internal load. METHODS: Ten healthy men performed two resistance training sessions (MS and P) which consisted of three exercises (bench press, peck deck and decline bench press) with the same total volume of load lifted. The training sessions were performed 14 days apart and allocated in a counter-balanced order. Hormonal (plasma insulin, growth hormone [GH], testosterone and cortisol) and metabolic (blood glucose and lactate) responses were assessed before and after each exercise bout. Session rating of perceived exertion (session RPE) was taken 30-min following each bout. RESULTS: No difference was observed for session-RPE between P and MS bouts (P>0.05). Plasma GH, cortisol and lactate increased significantly after exercise both bouts (P<0.01), but there were no significant changes between MS and P (P>0.05). CONCLUSION: It is concluded that the acute bout of resistance exercise following MS and P systems provide similar training strain when the total volume of load lifted is matched.

Exercise-Based Strategies to Prevent Muscle Injury in Male Elite Footballers: An Expert-Led Delphi Survey of 21 Practitioners Belonging to 18 Teams from the Big-5 European Leagues.

PURPOSE: To define based on expert opinion and practical experience using a systematic and scientific approach, (1) the perceived most effective exercise-based strategies to prevent muscle injury in elite footballers; and, (2) when and how these exercise programs are prescribed based on the number of days between games i.e. implementation strategy. METHODS: A Delphi survey obtained opinions and assessed for agreement. Delphi respondents consisted of 21 experienced sports practitioners (12 ± 5.3 years in elite football and with an academic background) belonging to 18 teams from the Big-5 European football leagues; England, France, Germany, Italy, Spain. Three teams were represented collaboratively by two experts. The Delphi process involves sequential rounds each evolving based on the responses from the previous. The number of rounds is not pre-defined and continues until an agreement is either achieved or it is clear that no agreement will be reached. Frequency of responses was recorded where the agreement was sought (i.e. in closed questions) and an agreement was achieved if ≥ 13/18 (70%) respondents agreed. For open-ended questions, a qualitative content analysis was performed to identify recurring themes and when themes were specified by ≥ 13 (70%), these were also considered as reaching an agreement. Practitioners had the opportunity to raise concerns if they disagreed with the 'agreement from recurrent themes'. RESULTS: There were four Delphi rounds (100% response for each round). Sprinting and High-Speed Running (HSR) focused exercises were agreed as most effective (perceived) to prevent muscle injuries. Eccentric exercise was perceived as the next most effective. It was agreed that sprinting and HSR be integrated into coaches training, and target 100% of players worst-case match scenario (e.g. volume, intensity) based on individual maximum speeds. Eccentric exercise was recommended to be implemented according to the context of the main football session and planned/actual sprinting and HSR content. It was agreed that eccentrics can be performed before or after training, context dependent. The day to perform specific sprinting and HSR or eccentric exercises depended on the proximity of previous and upcoming matches. Other exercises reaching agreement as 'somewhat effective' included concentric and isometric, horizontal and vertical plyometrics, coordination, core and dynamic flexibility in addition to core stability. No agreement was reached for multi-joint, resisted sprinting, kicking or agility exercises nor simultaneous single-leg strength and stability. Finally, no agreement was reached regarding programming variables e.g. sets, repetitions as deemed too contextual. CONCLUSION: Regarding exercise-based strategies, particular importance agreed by the Delphi expert group was to focus on sprinting, HSR and eccentric exercises, integrated with a variety of other exercise modes which also carry some level of effectiveness in a multidimensional programme. Context was agreed to be key and decision-making about when to undertake/ how to prescribe exercise strategies to be made according to the content of normal football training and the proximity of matches.

Generic versus small-sided game training in soccer.

The aim of this study was to compare 7 weeks of soccer-specific small-sided game (SSG) and mixed generic fitness training, on selected physiological, perceptual and performance variables. Twenty-five elite youth players were randomly allocated to either a SSG (coach selected) or generic training group (GTG), in a randomised, parallel matched-group design. In addition to normal training, each group completed two fitness training sessions per week of equal duration. Players completed a V O (2 max) treadmill test, Multistage Fitness Test (MSFT), Yo-Yo Intermittent Recovery Test Level 1 (YYIRTL1), 12x20 m test of repeated-sprint ability (RSA) and 20-m sprint test pre and post training. Training heart rate, perceived training intensity and perceptual fatigue measures were recorded throughout the training period. There were no differences in training heart rate or perceptual well-being measures. However, the GTG did perceive their training to be more intense than SSG. There were no changes in either group for V O (2 max), MSFT, RSA or sprint performance. However, there were improvements in YYIRTL1 performance for both groups over time, but not between groups. The results show that both types of training are equally effective at improving pre-season YYIRTL1 performance, despite GTG being perceived to be more intense.

The reliability of the i-STAT clinical portable analyser.

The purpose of this investigation was to assess the reliability of the i-STAT clinical portable analyser and CG(4)(+) cartridge measures of blood pH, partial pressures of O(2) (pO(2)) and CO(2) (pCO(2)), bicarbonate ([HCO(3)(-)]), base excess (BE), total carbon dioxide (TCO(2)), oxygen saturation (sO(2)) and blood lactate ([BLa(-)]) at various exercise intensities. A comparison between [BLa(-)] measured by the i-STAT and the Accusport lactate analysers during an intermittent treadmill run was also undertaken. The technical error of measurement (TEM%) at rest, at moderate (200W) and maximal exercise (V O(2)max) was acceptable (<15%) for all parameters. The intra-class correlation coefficients for each analyte ranged from weak-to-strong across resting (ICC=0.19-0.96) and moderate (ICC=0.30-0.96) exercise intensities. The ICC for all analytes were observed to be strong following maximal intensity exercise (ICC=0.77-0.95). The comparison of the [BLa(-)] measures between the i-STAT and Accusport showed that the difference between measures was acceptable at both low (<4mmolL(-1)) (-0.39+/-0.27mmolL(-1)), moderate to high concentrations (>4mmolL(-1)) (0.58+/-1.22mmolL(-1)), and across all [BLa(-)] data (0.36+/-1.13mmolL(-1)). In conclusion, the i-STAT clinical analyser and CG(4)(+) cartridge provides reliable measures of a number of blood parameters across exercise intensities. The [BLa(-)] measures from the i-STAT analyser are consistent with that of the Accusport lactate analyser.

Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells.

The mitogen-activated protein kinase (MAPK) signal transduction pathway plays an essential role in cell cycle progression and can be activated by many growth factor/mitogen pathways including estrogen. MAPK has also been implicated in ligand-independent activation of estrogen receptor-alpha (ER-alpha). The development of estrogen-independent growth in breast cancer is likely a first step in progression to hormone independence and antiestrogen resistance. We examined MAPK expression and activity in T5-PRF and T5 human breast cancer cells. T5-PRF is an estrogen-nonresponsive cell line developed from T5 cells by chronically depleting the cells of estrogen in long-term culture. MAPK activity measured in vitro was significantly higher (P < 0.05) in T5-PRF compared with T5 cells. Western blot analyses showed increased levels of active dually phosphorylated MAPK in T5-PRF cell extracts compared with T5. The increased activity and expression of MAPK may contribute to the estrogen nonresponsive growth phenotype and ligand-independent activity of ER in T5-PRF cells.

Agonist-induced internalization and trafficking of cannabinoid CB1 receptors in hippocampal neurons.

Agonist-induced internalization of G-protein-coupled receptors is an important mechanism for regulating receptor abundance and availability at the plasma membrane. In this study we have used immunolabeling techniques and confocal microscopy to investigate agonist-induced internalization and trafficking of CB(1) receptors in rat cultured hippocampal neurons. The levels of cell surface CB(1) receptor immunoreactivity associated with presynaptic GABAergic terminals decreased markedly (by up to 84%) after exposure to the cannabinoid agonist (+)-WIN55212, in a concentration-dependent (0.1-1 microm) and stereoselective manner. Inhibition was maximal at 16 hr and abolished in the presence of SR141716A, a selective CB(1) receptor antagonist. Methanandamide (an analog of an endogenous cannabinoid, anandamide) also reduced cell surface labeling (by 43% at 1 microm). Differential labeling of cell surface and intracellular pools of receptor demonstrated that the reduction in cell surface immunoreactivity reflects agonist-induced internalization and suggests that the internalized CB(1) receptors are translocated toward the soma. The internalization process did not require activated G-protein alpha(i) or alpha(o) subunits. A different pattern of cell surface CB(1) receptor expression was observed using an undifferentiated F-11 cell line, which had pronounced somatic labeling. In these cells substantial CB(1) receptor internalization was also observed after exposure to (+)-WIN55212 (1 microm) for relatively short periods (30 min) of agonist exposure. In summary, this dynamic modulation of CB(1) receptor expression may play an important role in the development of cannabinoid tolerance in the CNS. Agonist-induced internalization at presynaptic terminals has important implications for the modulatory effects of G-protein-coupled receptors on neurotransmitter release.