Effect of creatine supplementation on intramuscular TCr, metabolism and performance during intermittent, supramaximal exercise in humans.

This study examined the effect of (a) creatine supplementation on exercise metabolism and performance and (b) changes in intramuscular total creatine stores following a 5 day supplementation period and a 28 day wash-out period. Six men performed four exercise trials, each consisting of four 1 min cycling bouts, punctuated by 1 min of rest followed by a fifth bout to fatigue, all at a workload estimated to require 115 or 125% VO2,max. After three familiarization trials, one trial was conducted following a creatine monohydrate supplementation protocol (CREAT); the other after 28 d without creatine supplementation, in which the last 5 d involved placebo ingestion (CON). Intramuscular TCr was elevated (P < 0.05) in CREAT compared with the final familiarization trial (FAM 3) and CON. Concentrations of this metabolite in these latter trials were not different. In addition, a main effect (P < 0.05) for treatment was observed for PCr when the data from CREAT were compared with CON. In contrast, no differences were observed in the total adenine nucleotide pool (ATP+ADP+AMP), inosine 5'-monophosphate, ammonia, lactate or glycogen when comparing CREAT with CON. Despite the differences in TCr and PCr concentrations when comparing CREAT with other trials, no difference was observed in exercise duration in the fifth work bout. These data demonstrate that creatine supplementation results in an increase in TCr but this has no effect on performance during exercise of this nature, where the creatine kinase system is not the principal energy supplier. In addition 28 d without supplementation is a sufficient time to return intramuscular TCr stores to basal levels.

Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release L-dopa in vitro.

Two PC12 cell-derived lines have been studied following encapsulation into polymer-based hollow fibers with respect to secreted catecholamines and their metabolites. Cellular encapsulation provides a chronic microperfusion environment within which basally secreted PC12 products can be readily measured. Encapsulated PC12 cells grown and held under the conditions specified in this report basally release amounts exceeding their total cellular stores of the dopamine precursor L-DOPA and the electrochemically active dopamine metabolites DOPAC and HVA during 45-min static incubations. Under these same conditions, these cells release less than 0.1% of their total cellular store of dopamine. Depolarizing incubations enhance dopamine secretion eightyfold and enhance secretion of L-DOPA, HVA, and DOPAC about twofold. The relative composition of products basally secreted differs between PC12-derived cell lines, and an inverse relationship exists between basal release of L-DOPA and total cellular store of dopamine. These results further indicate that selected PC12 cell lines are potentially a source of both dopamine and L-DOPA in therapeutic cellular replacement applications.

Regeneration of the neurohemal terminals for identified cerebral neurosecretory cells in an insect.

The axons of specific neurosecretory cells, L-NSC III, in the brain of the tobacco hornworm, Manduca sexta, were transected during larval-pupal development to study the effects of this type of lesion on these peptidergic neurons and to begin to identify factors that may regulate their regeneration and growth. The two somata of these bilaterally paired neurons produce the prothoracicotropic hormone and are located in the pars intercerebralis. Their axons exit from the contralateral brain lobe via a retrocerebral nerve and pass through the corpus cardiacum before terminating at the glandular corpus allatum. At the corpus allatum, the L-NSC III axons arborize to form the terminal neurohemal organ for prothoracicotropic hormone release. The retrocerebral nerve was severed either in vitro followed by brain transplantation or in situ; in either protocol, the distal axon segments and corpus allatum were removed. The ability of the injured L-NSC III axons to regenerate was assessed immunocytologically by using a monoclonal antibody against the prothoracicotropic hormone. In both treatments, the proximal axon stumps exhibited regenerative growth as early as 1 day after axotomy, and, by the third day, neurites had extended. By the fifth day, the regenerating axons had branched to form terminal varicosities similar to those of a normal neurohemal organ. The regenerated neurohemal structure appeared to be functional, because larvae that had been bilaterally axotomized were able to metamorphose to pupae, a process requiring temporally precise periods of prothoracicotropic hormone release. In addition to the regeneration of the terminal axon structures, several other responses to axotomy and retrocerebral organ excision occurred. These included an apparent accumulation of prothoracicotropic hormone in the axons and regenerating neurohemal-like structure, sprouting of ectopic neurites from the axotomized somata, and a change in shape of the cell bodies from spherical to avoid.

A monoclonal antibody to the insect prothoracicotropic hormone.

The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.

Varicosity-associated antigens define neuropile subfields in the leech central nervous system.

A panel of twelve monoclonal antibodies raised against homogenates of leech nerve cords and four polyclonal antisera raised against purified neurotransmitters were used to label varicosities immunocytologically in the central neuropile of leech segmental ganglia. These immunoreactive varicosities occur in distinct patterns, some of which have a simple geometry. Three antibodies label immunoreactive varicosities distributed in a single dorsoventrally-oriented plane, two label varicosities distributed in lateral hemi-neuropiles (leaving void a central cephalocaudal passageway), and five label varicosities distributed throughout the neuropile. Six antibodies tested label varicosities across leech species, and five of these varicosity populations are distributed in patterns conserved across leech species. Immunocytologically-defined neuropile subfields do not correspond with previously identified histological and ultrastructural features of leech segmental ganglia. Analysis of immunocytologically-defined subfields is extended to include identification of sets of neurons which appear to project into these subfields, and to include an intracellular characterization of one of these neuron sets.

Cytological evidence for serotonin-containing fibers in an abdominal neurohemal organ in a hemipteran.

The abdominal neurohemal organs of the hemipteran Rhodnius prolixus contain an extensive serotonin containing arborization. Endogenous serotonin within fibers and terminals in the neurohemal area were detected with histochemical and immunocytological techniques. The abdominal nerves which contain the neurohemal organs selectively sequester exogenous serotonin. Serotonin and its metabolites are biochemically detected within the mesothoracic ganglion, which is a known source of projections into the neurohemal organ. However, the source of the cell bodies which might send serotonergic fibers to the neurohemal organ remains undetermined because no correspondence was found between immunocytological maps of serotonin-containing cells in the ganglion, and projection maps into neurohemal organ (determined by cobalt back-filling).

Serotonin modulation of the release of sequestered [3H]serotonin from nerve terminals in an insect neurohemal organ in vitro.

Exogenous tritiated serotonin ([3H]5-HT) is taken up by and released from serotonin-containing fibers within abdominal nerves of Rhodnius prolixus during in vitro incubations. Sequestered [3H]5-HT behaves as expected of an endogenous neurosecretory product in this system. Release is Ca2+-dependent during both K+-induced and physiologically induced secretory episodes. The kinetics of the release of sequestered label parallels the kinetics of release of endogenous neurohormones. Preloaded preparations which are washed with unlabeled 5-HT release label in two fashions; 5-HT itself induces a release of label and, at lower concentrations, 5-HT facilitates the release of label induced by high K+ washes. Facilitatory effects appear to be mediated through receptors within the neurohemal organ (NHO).