Effects of an inverted seated position on single and sustained isometric contractions and cardiovascular parameters of trained individuals.

Previous research demonstrated higher maximal voluntary contraction (MVC) force with upright vs. inverted positions in untrained individuals. The purpose was to determine the effects of inversion on force, activation, and cardiovascular responses before and following fatigue in trained individuals. Twelve male athletes completed two trials: upright and inverted seated positions. At baseline (upright), either leg extension (LE) or elbow flexion (EF) evoked contractile properties and MVCs were performed. LE and EF contractions were randomly allocated and performed in separate sessions. The subject was then positioned for 150s in each posture, followed by a 30s MVC (MVC30). During each trial, stroke volume (SV), cardiac output (Q), heart rate (HR), time and frequency domain HR variability measures and mean arterial blood pressure (MAP) measurements were recorded. ANOVA showed no statistical differences in EF MVC force, but a tendency (p=.12) for LE MVC decline with inversion vs. upright. Evoked resting (p=.1) and potentiated peak twitch (p=.04) force were increased with inverted LE but tended to diminish with inverted EF (p=.06 and p=.1). Force-fatigue, electromyography-fatigue relationships and HR variability during MVC30 fatigue were not affected. HR and Q were significantly (p=.01) lower with inversion following both LE and EF fatigue. Compared to the significant inversion-induced changes associated with untrained individuals in previously published studies, the lack of postural changes in resting force and CV measures may demonstrate that highly trained individuals adapt better to inversion.

Optimisation of platelet aggregometry utilising micotitreplate technology and integrated software.

The possible applications of the measurement of platelet aggregation by conventional photometry are limited by the short lifetime of platelet suspensions. Recently a microplate-based technique was introduced, which promised to revolutionize experimental design by enabling simultaneous assessment of 96 platelet samples. However, adoption of this technique has been slow since the available equipment was not able to satisfy all the conditions required for conventional aggregometry, especially adequate agitation of samples whilst maintaining total control of incubation temperature. This report describes how these problems may be overcome, together with the introduction of on-line analysis of data using commercially available software, obviating the complexities of data management previously encountered.

Protein phosphorylation in electrically permeabilized islets of Langerhans. Effects of Ca2+, cyclic AMP, a phorbol ester and noradrenaline.

The incorporation of 32P from [gamma-32P]ATP into intracellular proteins was studied in electrically permeabilized rat islets of Langerhans. Ca2+ (10 microM), cyclic AMP (100 microM) and a protein kinase C-activating phorbol ester, phorbol 13-myristate 12-acetate (PMA; 100 nM) produced marked changes in the phosphorylation state of a number of proteins in permeabilized islets after incubation for 1 min at 37 degrees C. Ca2+ modified the effects of cyclic AMP and PMA on protein phosphorylation. Noradrenaline (10 microM) had no detectable effects on Ca2+-dependent protein phosphorylation, but significantly inhibited Ca2+-induced insulin secretion from electrically permeabilized islets. These results suggest that electrically permeabilized islets offer a useful model in which to study rapid events in protein phosphorylation as a mechanism of stimulus-secretion coupling. If the rapid Ca2+-induced effects on protein phosphorylation are involved in the control of insulin secretion, the results of this study also imply that part of the catecholamine inhibition of insulin secretion occurs at a stage in the secretory pathway beyond the activation of the regulated protein kinases.

Early events in inositol phosphate metabolism in longitudinal smooth muscle from guinea-pig intestine stimulated with carbachol.

In longitudinal smooth muscle from guinea-pig intestine prelabelled with [3H]inositol, carbachol produced a 3-fold increase in [3H]inositol 1,4,5-trisphosphate within 2 s, and there was also a simultaneous increase in [3H]inositol 1,4-bisphosphate. 3H-labelling of inositol 1,3,4,5-tetrakisphosphate was not significantly increased until 60 s after carbachol stimulation, and the accumulation of [3H]inositol 1,3,4-trisphosphate was relatively small.

Calcium channels and calcium-mediated effects in smooth muscle cells.

In single smooth muscle cells held at depolarized potentials under voltage-clamp, spontaneous transient outward currents (STOCs) of about 100 ms in duration and 100-300 pA in size are observed. These behave as if they are caused by the cyclical release of calcium from storage sites within the cell and can therefore be used as a method of monitoring the release of calcium from these stores by various agents. Muscarinic receptor activation or caffeine applied to single smooth muscle cells from rabbit jejunum produced outward current, probably due to the release of calcium from stores, and STOCs were subsequently abolished. Levels of inositol 1,4,5 trisphosphate in fragments of the muscle were increased within 5 s by carbachol application. STOCs and outward current due to calcium store release were probably due to the opening of calcium-activated K-channels since they were abolished by 4 mM tetraethylammonium. Receptor activation also opens channels that admit cations, including calcium, which may also contribute to tension generation.

Decreased responsiveness to chronic salmon calcitonin treatment in rat kidney and calvaria studied using quantitative enzyme cytochemistry.

Growing rats were treated with daily im doses of salmon calcitonin (sCT) (2, 15 and 100 IU/kg) for various times (1, 4 and 24 weeks). The effects on intracellular enzyme activities in bone and kidney were monitored using quantitative cytochemical methods previously developed for the identification of specific target tissue responses to calcitonins. The basal alkaline phosphatase activities in both kidney and bone were decreased by long-term treatment at all time periods and doses tested. No change was noted in basal Ca ATPase activities in kidney after treatment. The capacity of target tissues in chronically treated and control rats to respond to an acute iv dose of sCT was also compared. Acute provocation tests in treated and control rats showed that the renal alkaline phosphatase response was decreased in the rats receiving long-term treatment. Moreover, the direction of response was reversed in chronically treated rats when bone alkaline phosphatase and renal Ca-dependent ATPase activity was measured after acute provocation with sCT, i.e. bone alkaline phosphatase was stimulated instead of being inhibited and renal Ca ATPase was inhibited instead of being stimulated. The application of quantitative cytochemical techniques has demonstrated intracellular changes in enzyme activities in both kidney and bone. The impaired sCT responsiveness can be detected at shorter times of treatment (1 week) and lower doses (2 IU/kg) than has previously been possible by measurement of indices of mineral metabolism in plasma or urine.

Effect of dietary fat content on the incidence of obesity among ad libitum fed mice.

Graded increments in the fat-to-carbohydrate ratio of the diet elicited a gradual, but reversible increase in the average mass of body fat maintained by adult female (CDI) albino mice under ad libitum feeding conditions. In addition, the inter-individual variability in the animals' fat mass was greatly magnified by diets with a substantial fat content (greater than 30 percent of calories). As a result, the incidence of obesity (defined as one third or more of body weight as fat) increased progressively from 0 percent to 35 percent when the diet's fat content was varied from 1 percent to 64 percent of its total energy content. A state of weight maintenance can only become established when the relative rates of glucose and fatty acid oxidation are proportional, on average, to the carbohydrate-to-fat ratio of the diet. When diets with a relatively high fat content are consumed, a considerable enlargement of the adipose tissue mass appears to be necessary in many animals before weight maintenance becomes spontaneously established. It is proposed, therefore, that changes in the adipose tissue mass, along with shifts in the range in which glycogen levels are maintained, and other adaptive changes, contribute to bring about rates of fat oxidation commensurate with a diet's fat content. This impact of dietary composition on body composition may be a factor contributing to the increased incidence of obesity in affluent populations consuming diets with a substantial fat content.

A dual pathway for ACTH steroidogenic action in purified adrenocortical cells.

Isolated adrenal fasciculata cells were purified by centrifugation through a 0-50% hyperbolic gradient of PercollR. The dose-dependence and kinetics of both intracellular cyclic AMP accumulation and steroidogenesis in response to ACTH1-39 and ACTH5-24 (corticotropin-(1-39) and corticotropin-(5-24)-peptides) were determined using purified cells. The rate of intracellular cyclic AMP formation was maximal during the first five minutes after hormone addition and remained constant or fell thereafter. Therefore intracellular cyclic AMP accumulation, assessed after 5 min., was compared with steroid output after 20 min. Maximal steroidogenesis was elicited by ACTH5-24 without discerning a significant stimulation of intracellular cyclic AMP accumulation. ACTH6-24 (corticotropin-(6-24)-peptide) could completely inhibit the intracellular cyclic AMP accumulation elicited by ACTH1-39 or by ACTH5-24 at concentrations that only partially inhibited steroidogenesis. It is possible that there are two pathways for the steroidogenic action of ACTH, one of which is obligatorily mediated by intracellular cyclic AMP, and another which involves a different mediator.

Quantitative cytochemical responses to exogenously administered calcitonins in rat kidney and bone cells.

Time- and dose-dependent changes in intracellular enzyme activities in kidney and bone from rats injected with calcitonin have been assessed by quantitative cytochemistry. The doses of salmon calcitonin given were similar to those suggested in the Pharmacopoeial rat hypocalcaemia bioassay (1-50 mIU/50 g body weight). The highest doses produced 30% inhibition of alkaline phosphatase activity, maximal within 20 min after injection, in cells of renal proximal tubules and a stimulation of calcium-dependent adenosine triphosphatase activity in kidney cortical and outer medullary cells. Alkaline phosphatase activity in the periosteal bone cells was markedly inhibited at the lowest doses. When doses of human and porcine calcitonins were given which would be equipotent with that of salmon calcitonin in the rat hypocalcaemia bioassay, the effect of the non-mammalian peptide on renal alkaline phosphatase activity was relatively greater than that of the mammalian peptides. Oxidized human calcitonin did not inhibit renal alkaline phosphatase activity even when an amount equivalent to 10 times the highest dose of the unmodified peptide was injected.

Proposed mechanism of cholinergic action in smooth muscle.

An increased turnover of phosphatidate and phosphatidyl inositol has been found in many tissues where hormones or neurotransmitters are postulated to raise Ca2+ influx, for example in smooth muscle. However, the relationship between changes in phospholipid metabolism and changes in Ca2+ permeability was unknown. Following recent reports on the interactions of Ca2+ with phosphatidic acid in membranes and artificial systems, we investigated the hypothesis that phosphatidate accumulation mediates the action of cholinergic and other stimuli on Ca2+ influx. We report here that synthesis and accumulation of phosphatidate was accelerated in smooth muscle cells stimulated by carbamylcholine with a similar time course to that of contraction. This alteration in phosphatidate metabolism does not seem to result from an increase in intracellular Ca2+ or depolarisation of the cell membrane. Furthermore, submicromolar concentrations of phosphatidate rapidly produce contractions of isolated smooth muscle cells. These results support the contention that cholinergic-induced changes in membrane Ca2+ permeability in smooth muscle could be mediated by phosphatidate accumulation.

Phosphatidic acid inhibition of PGE1-stimulated cAMP accumulation in WI-38 fibroblasts: similarities with carbachol inhibition.

To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.