Intracellular calcium release mediated by sphingosine derivatives generated in cells.

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.

Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels.

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Perceived barriers to physical activity across Norwegian adult age groups, gender and stages of change.

This study investigated differences in the type of and number of perceived barriers to engagement in physical activity experienced by adult women and men in the same geographical area, the relationship between the experienced barriers and stages of change in relation to exercise behavior, and identified barriers related to reported engagement in leisure-time physical activity. Data were obtained from a population study by the National Institute of Public Health in two counties during 2000-2001. The sample consisted of 2709 females and 2212 men in the age groups 75, 60, 45, 40 and 30 years. Questionnaires measured barriers to engagement in physical activity, engagement in physical activity and readiness for engaging in physical activity (stages of change). Multivariate analyses of variance demonstrated significant age and gender differences in the perceptions of barriers at the various stages of change. The logistic regressions [estimated odds ratios (OR)] demonstrated that low scores for affective/cognitive and practical barriers were significantly associated with higher OR for engagement in physical activity for women, and low-priority barriers and lower age were associated with higher OR for being physically active for men. The information from this study should be valuable for designing and tailoring both motivational strategies and interventions to fit targeted groups.

Evaluation of an exercise programme for post-bariatric surgery patients: views of participants.

OBJECTIVE: Exercise programmes typically are evaluated with fitness assessments and psychological survey measures but seldom include participants' insights. The purpose of this study was to evaluate the benefits, facilitators and barriers of a 12-week exercise programme for post-bariatric surgery patients from the participants' perspective. METHOD: Over a 2-year period, 20 patients recently having undergone bariatric surgery completed a 12-week programme that included participation in structured exercise and in focus groups designed to supplement standard evaluation data and provide insight into participants' views. RESULTS: Participants were highly adherent to the programme, and focus group results reflected a clear positive evaluations. Benefits included helpful information, developing commitment, physical fitness and social support; notably, weight was seldom mentioned. Participants cited structure, accountability and group support as facilitators of exercise. Participants cited few barriers, although very few had set plans for continuing exercise after programme completion. CONCLUSION: Participants saw many benefits to the exercise programme, and those benefits reflected lifestyle changes rather than a focus on weight. Programme structure, accountability and the support of the group were facilitators to exercise. Participants cited few barriers. However, the lack of plans for continued exercise suggested the need for a transition phase to help participants continue an active lifestyle after the 12-week structured programme.

TRPC channels: integrators of multiple cellular signals.

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.

The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin.

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase family of intracellular Ca(2+)-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca(2+)-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Cai) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Cai is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian M(r) 28,000 calbindin D Ca(2+)-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (> 1 h) elevation in Cai and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., > 1 h) elevation in Cai.

A common mechanism underlies vertebrate calcium signaling and Drosophila phototransduction.

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP(3)R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP(3) branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis we applied the membrane-permeant InsP(3)R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP(3)R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP(3)-mediated Ca(2+) release and store-operated Ca(2+) entry in Xenopus oocytes at a stage operating after production of InsP(3) but before the opening of the surface membrane Cl(-) channels by Ca(2+). We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP(3) branch of the inositol lipid-signaling pathway.

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities.

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.

Calcium signalling mechanisms in endoplasmic reticulum activated by inositol 1,4,5-trisphosphate and GTP.

Ca2+ signals are known to mediate an array of cellular functions including secretion, contraction, and conductivity changes. In spite of the obvious role of Ca2+ in signalling, the control of Ca2+ within cells is known to be a complex phenomenon involving a number of distinct active and passive transport systems functioning within different organelles. Inositol 1,4,5-trisphosphate (IP3) is now established as a central mediator of Ca2+ mobilization, and the endoplasmic reticulum (ER) has been considered to be the site of action of IP3. However, this role has been ascribed almost by default to the ER, based on the knowledge that IP3 functions to release Ca2+ from an intracellular, nonmitochondrial, Ca2+-pumping organelle. Our interest has been to ascertain by what mechanism IP3 activates Ca2+ movements, at what intracellular locations it functions, and how the size and replenishment of the IP3-sensitive Ca2+ pool occurs. During the course of such studies, another mechanism inducing profound movements of Ca2+ within cells was identified. This process is activated by a highly sensitive and specific guanine nucleotide regulatory mechanism, which, while inducing fluxes of Ca2+ that resemble the action of IP3 under certain conditions, has now been determined to involve a quite distinct mechanism. The characteristics of this mechanism are described below. Although involving a very different Ca2+ translocation process to that activated by IP3, several important conclusions have been drawn on the relationship between IP3- and GTP-activated Ca2+ movements leading us to believe that the latter may have a regulatory role in controlling the size and/or entry of Ca2+ into the IP3-sensitive Ca2+ pool.

Effects of immunoglobulins upon the interaction of thyrotropin with receptor in human fat tissue.

TSH bound to human fat membranes is shown to be unusually sensitive to the addition of immunoglobulin G (IgG). Significant displacement was observed with 10 microgram/ml, and 50% displacement was obtained with 50-100 microgram/ml IgG prepared from control sera pooled from euthyroid subjects. It was not possible to demonstrate increased TSH displacement by IgG preparations from sera of thyrotoxic compared to that of euthyroid subjects using human fat membranes, as was demonstrable with human thyroid membranes. The increased sensitivity of fat membranes to IgG was not due to nonspecific protein interactions and is suggested to derive from interactions with Fc receptors associated with fat membranes.

Characterization of thyrotropin binding to specific receptors in human fat tissue.

Specific receptors for thyrotropin were found to exist in membranes from whole human subcutaneous fat tissue. The characteristics of the interaction of 125I-labelled thyrotropin with such receptors were determined and compared with the stable, high-affinity thyrotropin receptor shown previously to exist in guinea pig fat membranes. Specific binding was readily detectable using low concentrations of membranes (up to 80 microgram/ml), though specific binding was reduced at higher membrane concentrations. Increasing concentrations of unlabelled thyrotropin reduced fractional binding, revealing saturation of a population of mixed affinity sites (highest Ka of the order of 0.3 X 10(9) M-1). Little cross-reactivity was observed with other lipolytic or structurally related hormones, though some cross-reactivity was observed in the presence of human chorionic gonadotropin. Association was temperature-dependent and rapid at 37 degrees C, though prolonged incubation revealed some instability of binding at this temperature. Binding was reversible with a high dissociation rate constant, and was particularly sensitive to the presence of low concentrations of sodium or calcium ions. Using membranes prepared from isolated human fat cells, binding of thyrotropin was equally sensitive to the addition of cations.

Calcium pools, calcium entry, and cell growth.

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluorescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following Insp3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca(2+)-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G(o)-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells re-enter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

Knowledge of results precision and motor skill acquisition.

The effect of varying KR precision levels on the initial acquisition and later performance of a linear positioning task was investigated with 40 female undergraduates serving as subjects. Subjective evaluations of performance and estimates of accuracy were recorded along with actual scores. KR precision level (centimeters or millimeters) did not affect actual performance, but extremely precise KR (millimeters) had detrimental effects on performance evaluations and the labeling process of estimate performance.

The prediction of group motor performance from individual member abilities.

Two experiments were conducted to investigate the prediction of group performance on the motor-maze task from individual member abilities. In both experiments subjects performed 20 individual trials on the maze task in an initial session prior to the actual experiment. On the basis of these individual performance scores, two-person groups were formed so that the groups represented a range of average ability levels and a range of discrepancies in abilities between partners. In Experiment 1, all groups performed a cooperative and a noncooperative group task in separate sessions both involving competition with another group. Only the cooperative group task was used in Experiment 2, but all groups performed under both competitive and noncompetitive conditions. Multiple regression analyses yielded a moderate, positive relationship between member abilities and group performance in both experiments; group performance on the highly cooperative task was dominated by the lower-ability partner.

The informational and motivational influence of social reinforcement on motor performance.

Two experiments were conducted to determine the effects of social reinforcement on the performance of a motor task. Subjects for both experiments were seventh-and eighth-grade girls from a public junior high school. Experiment 1 examined the influence of knowledge of results (KR) and social reinforcement on 25 initial acquisition trials of a novel motor task in a KR × social reinforcement × experimenter blocks (2 × 2 × 2 × 5) factorial design. Experiment 2 examined the influence of KR and social reinforcement on 40 later performance trials in a KR × social reinforcement × blocks (2 × 2 × 8) factorial design. Actual and estimated performance scores were analyzed with MANOVA, discriminant function analyses, and step-down F tests. Experiment 1 suggested a combination of informational and motivational effects, with both KR and social reinforcement facilitating performance. Social reinforcement did not significantly affect performance in Experiment 2.

Strengthening physical self-perceptions through exercise.

Two studies examined the effects of physical activity/exercise on physical self-perceptions, self-efficacy, body satisfaction, fitness and relationships among these variables. In study 1, 34 female undergraduates participated in a 10-week exercise/activity program. Participants were selected from existing classes forming a weight training, aerobic exercise and activity control group. Results revealed changes in physical self-perceptions, strength, and body composition over the 10-weeks. Improvements in physical self-perceptions and fitness occurred independent of exercise/activity group. Groups differed in the perceived importance attached to physical self-perceptions. Correlations among the measures revealed relationships among physical self-perceptions, body satisfaction, global self-esteem, and fitness. In study 2, we hypothesized that weight training would have a greater effect on physical self-perceptions and body image perceptions than physical education activity classes. Thirty-seven males and 28 females were selected from existing classes forming a weight training and activity group. Results revealed no significant changes in physical self-perceptions, body image, or global self-esteem over the 10-week program, while strength and physical self-efficacy improved. Correlations among measures from both studies offer preliminary support for Sonstroem and Morgan's model for the examination of self-esteem in exercise settings.

Competitive trait anxiety, success-failure and sex as determinants of motor performance.

Two experiments determined the effects of competitive trait anxiety, success-failure, and sex on the performance of 10- to 12 yr.-old children competing on a complex motor maze. Competitive trait anxiety was assessed by the Sport Competition Anxiety Test and success-failure was induced by giving bogus win-loss feedback. High and low competitive trait-anxiety children were randomly assigned to one of three conditions: winning 80%, 50% or 20% of 20 contests. The average completion time and the variability of times within each of two blocks of 10 contests were the two performance measures. State-anxiety was assessed with Spielberger's State Anxiety Inventory for Children as an indicant of arousal prior to and during competition. The findings of Exp. 1 yielded no significant performance differences. In Exp. 2 a significant interaction of competitive trait anxiety X success-failure X sex for performance time and variability was obtained. This interaction was largely attributed to sex differences.