The nociceptive component of the flexion reflex (FR) has been observed by several investigators to be a reliable physiological manifestation of pain perception in man. It has thus been used as an index of spinal nociceptive activity to study analgesic mechanisms. Vibration is one analgesic procedure which has been reported to decrease subjective pain report in certain pain conditions. The objective of this study was thus to quantitatively investigate the behavior of the human FR during segmental peripheral conditioning with high-frequency vibration. / A first study was undertaken to determine the relationship between pain sensation and the FR under the paradigm developed in this laboratory. Visual analogue scale (VAS) ratings of subjective sensation were found to bear a high, linear correlation with stimulus intensity (r = 0.95) and with FR area (r = 0.91). / In the main study, we found segmental vibration to inhibit the FR in 5 of 9 subjects. The modulatory effects displayed a slow onset and decay, lasting beyond the 30 min vibration period. Maximum modulation occurred late into vibration, and in some cases after vibration. The direction and magnitude of modulatory effects were basically the same in both biceps femoris (BF) and tibialis anterior (TA) muscles. Our results raised the possibility that prolonged vibration could influence spinal nociceptive reflexes in ways similar to the vibration-induced pain relief described in the clinical literature.

Post-inspiratory braking is the activation of inspiratory muscles during expiration. Although not energetically optimal, it exists for currently unexplained reasons. It has been postulated that post-inspiratory braking exists in order to maintain arterial CO2 content in a tightly regulated band. A computer simulation of the respiratory system incorporating respiratory control, respiratory mechanics, and gas exchange dynamics was developed in order to evaluate the relationship of post-inspiratory braking to arterial CO 2 content. The simulation was driven with pressure signals having varying amounts of post-inspiratory braking. No significant effect upon arterial CO 2 content was found. This suggests that post-inspiratory braking does not exist for the purposes of increasing CO2 exchange in the lungs.

The present study examines the distribution of the low density lipoprotein receptor related protein-2 (LRP-2) in the reproductive tracts of male and female rats. LRP-2 has been shown to facilitate the endocytosis, and lysosomal degradation of the glycoprotein SGP-2 in cultured cells. SGP-2 has been found in the male reproductive tract, is thought to be involved in sperm maturation. The apical surfaces of the epithelia of the efferent ducts as well as the intermediate zone, proximal caput, corpus, and cauda regions of the epididymis showed pronounced peroxidase staining for LRP-2. Immunogold labeling representing LRP-2 was observed in endocytic vesicles and early endosomes in the single labeling experiment. Labeling was much more significant in the efferent ducts than the epididymis. The double labeling experiment for LRP-2 and SGP-2 showed colocalization in the endocytic vesicles and early endosomes. SGP-2 also labeled the late endosomes and lysosomes. These results suggest that, LRP-2 mediates the endocytosis of SGP-2, and afterward is recycled back to the cell surface. The second aspect of the study examines the distribution of LRP-2 and SGP-2 in the epithelium of the uterus and the oviduct, in relation to the different stages of the estrus cycle. The surface epithelium and the epithelial cells lining the glands of the uterus were for the most part devoid of staining for LRP-2, except in the estrus stage when a strong reaction was seen in the apical region of the cells. The epithelium lining the glands of the uterus showed strong staining, while weak immunoperoxidase staining was observed for SGP-2 in the uterine surface epithelium in every stage of the estrus cycle. The epithelium of the proximal and distal, oviduct showed very little staining if at all for LRP-2 and SGP-2 for all stages of the estrus cycle. (Abstract shortened by UMI.)

Intracellular calcium has been widely demonstrated as an important part of intracellular signalling pathways. Entry of calcium in cells has been shown to trigger a variety of processes such as contraction in smooth and skeletal muscle, release of neurotransmitter in neurons and activation of modifying proteins in most cells, which in turn will activate or deactivate other processes. For these reasons, calcium permeation through any ion-channel type, whether ligand or voltage-gated, expressed on the surface of a cell, might potentially influence the intracellular activities or status of that cell. Calcium permeation in muscle nicotinic acetylcholine receptors has long been shown unequivocally. Calcium permeation through neuronal nAChR's has also been shown, but these experiments were done on preparations of neurons. Typically, neurons might express two or three subunits of the same type. The lack of knowledge on the assembly of subunits into receptors and the possibility that subunits might be permutable in a receptor make the exact subunit composition of the receptors unknown. To test the hypothesis that calcium is a permeant ion of the alpha 4beta2 subtype neuronal nicotinic acetylcholine receptor (nAChR), I performed single-channel patch-clamp experiments on micro-injected Xenopus oocytes expressing alpha4beta2 neuronal nAChR. When recording from single cell receptors in increasing concentration of extracellular Ca2+ I found that the amplitude of the single-channel current decreased with each increment in concentration. The behaviour of the resulting current-voltage relationships is described as a direct influence of calcium permeation on single-channel current amplitude. Analysis with a theoretical model describing permeation at the single-channel level supported this conclusion.

The measurement and analysis of the human diaphragm electromyogram (EMGdi), as obtained with an esophageal electrode, requires objective control of the disturbances and filtering effects which can influence the signal. One issue of importance is that an increase in the muscle-to-electrode distance (MEdist) acts as a low-pass filter, filtering out the high frequency components of the EMG power spectrum (the MEdist filter). Due to the numerous factors which can influence the EMGdi, control of signal quality is also of utmost importance. The aims of this study were: (1) to evaluate the effect of the MEdist filter on EMGdi, as measured with a multiple array esophageal electrode, (2) to take advantage of the MEdist filter in order to locate the position of the diaphragm with respect to the electrodes, and (3) to evaluate the influence of changes in chest wall configuration on EMGdi center frequency (CF) values, while controlling for both signal quality and the MEdist filter. / Five normal male subjects performed static contractions of the diaphragm at seven predetermined chest wall configurations. The EMGdi was measured with an array of eight steel rings mounted on a catheter, forming seven sequential pairs of electrodes, with an interelectrode distance of 10 mm. EMGdi signal quality was evaluated by computer algorithms. The pair of electrodes whose EMGdi signals (and power spectrums) were the least influenced by the MEdist filter was assumed to be closest to the diaphragm. / The results of the study indicated (1) EMGdi power spectrums and their associated CF values were strongly affected by the position of the diaphragm with respect to the multiple array esophageal electrode. CF decreased by approximately 1 Hz per mm displacement away from the diaphragm. (2) By controlling for the MEdist filter, there was no relationship found between changes in chest wall configuration and CF values. These data demonstrate that changes in chest wall configuration, and hence diaphragm length, do not influence the CF values of the EMGdi, if the distance between the electrodes and the diaphragm and signal quality are controlled for.

What determines the amplitude and the time course of spontaneous excitatory unitary events is not well understood. Desensitization of fast AMPA/K channels has been shown to be fast enough to shorten individual quantal responses. However, it is not known whether the desensitization is uniform nor is it known what determines the relation between amplitude and rise or decay times in absence of desensitization. Both the rise and the decay times are essentially amplitude-independent in control conditions, but become markedly amplitude-dependent after desensitization has been suppressed by aniracetam. Strong amplitude-dependence of the rise and decay times suggests that in absence of desensitization the process(es) that determine(s) the time course of unitary events is/are concentration-dependent. Highly nonuniform, and strongly amplitude-dependent desensitization can be explained by its concentration dependence. The scatter of both rise and decay times about the best fitting lines was also amplitude-independent in control conditions. It increased markedly and in an amplitude-dependent manner after addition of aniracetam. The same factors that determine mean values probably determine the scatter as well.

Intercellular junctions within the mammalian testes play an important role during spermatogenesis. The cadherins are a superfamily of integral membrane proteins that mediate calcium-dependent cell adhesion and are involved in the formation of intercellular junctions. The cadherin subtype, known as N-cadherin, mediates the interaction between the germ cells and Sertoli cells of the testis. Previous studies have shown that estrogens are able to modulate N-cadherin mRNA levels in the 7-day-old mouse testis. / I have examined the estrogen effects on N-cadherin protein levels in the testes of 21-day-old rats. The rats were injected with either 17$ beta$-estradiol, the anti-estrogens Tamoxifen and ICI 182,780, or estradiol in combination with ICI 182,780. Immunoblotting analysis of testicular proteins shows that N-cadherin levels in the 21-day-old rat are stimulated by estrogen. Treatment with anti-estrogens decrease N-cadherin levels. Administration of estrogen was able to overcome the inhibitory effects of anti-estrogen treatment. The results obtained from these studies indicate that the effect of estrogen on testicular N-cadherin levels is mediated through the estrogen receptor. Estrogen receptor levels within the testis are not altered by exogenous estrogen, but can be affected by treatment with anti-estrogen. / These experiments support the hypothesis that estrogens play an important role in spermatogenesis. In addition, the results provide insight into how disruption of testicular estrogen responsiveness can reduce fertility and why chronic anti-estrogen treatment results in disorganization of the seminiferous epithelium.

Activation of GABA$ rm sb{B}$ receptors inhibits spontaneous and evoked neuronal activity in different regions of the central nervous system, including the nucleus tractus solitarius (NTS), the projection site of pulmonary vagal afferent fibres. These studies were designed to test the hypothesis that GABA$ rm sb{B}$ receptors in the NTS modulate the vagally mediated cardiorespiratory reflexes. / Experiments were conducted on 53 spontaneously breathing, urethane-anesthetized (1.2-1.6 g/kg) and atropinized (0.2 mg/kg) rats. Tracheal pressure, tidal volume, mean arterial blood pressure, and the integrated diaphragmatic electromyogram (DiEMG) were recorded. The medulla was prepared for bilateral NTS injection of 110 nl of saline (vehicle), and 60 pmol baclofen, and 2.8 nmol CGP35348, a GABA$ rm sb{B}$ receptor agonist and antagonist, respectively. Injection sites were confirmed histologically. / Results support the hypothesis, but suggest that the cardiorespiratory pattern and the vagally mediated reflexes are not tonically controlled by GABA$ rm sb{B}$ receptors under the present conditions. The different effects of baclofen on inspiratory time and expiratory time suggest a difference in the distribution of GABA$ rm sb{B}$ receptors in neuronal networks controlling each of these respiratory phases. (Abstract shortened by UMI.)

Cystic Fibrosis transmembrane conductance regulator (CFTR) Cl - channels are rapidly and efficiently deactivated by a type 2C serine/threonine phosphatase (PP2C). The efficiency of this regulation suggests that PP2C and CFTR are associated within a regulatory complex. Previous work has shown that PP2C and CFTR can be co-immunoprecipitated and reversibly coupled by exposing cell lysates to the hydrophilic cross-linker dithiobis [sulfosuccinimidyl proprionate]. While it is clear that PP2C can associate with CFTR, the nature of the interaction between them has not been defined. The goal of this study was to identify specific region(s) within CFTR that mediate this interaction. The interaction between CFTR and PP2C was strong enough to be reproduced in the absence of cross-linking agents, supporting its physiological relevance. We then translated CFTR in vitro and incubated it with cell lysates containing endogenous PP2C, however interaction between the proteins was not observed under these conditions. Since the association seemed to require some factor present in cells, a distal fragment of CFTR was expressed in vivo and used for pull down assays. PP2C was co-purified under these conditions, suggesting an interaction between PP2C and the C-Tail of CFTR. To further narrow down the region of interaction, three residues within the C-Tail (Y1424, LL1430/1431) that had been shown previously to interact with the AMP-activated protein kinase (AMPK, a known PP2C substrate and possible targeting protein) were mutated to alanines. When this mutant was used in a pull-down assay, no association with PP2C was observed, consistent with a role for AMPK-binding residues and AMPK in the association of PP2C with CFTR.

Reactive oxygen species (ROS) and nitric oxide (NO) modulate normal and septic skeletal muscle contractility and redox state. We evaluated the involvement of NO synthases (NOS) and heme oxygenases (HO) in nitrosative and oxidative stresses in sepsis-induced diaphragmatic contractile dysfunction. Sepsis was elicited by injecting rats and transgenic mice deficient in either inducible (iNOS) or neuronal (nNOS) isoforms with 20 mg/kg E. coli lipolysaccharide (LPS). Peroxynitrite formation was monitored by measuring nitrotyrosine immunoreactivity with selective antibodies. Oxidative stress was assessed by measuring protein oxidation (carbonyl groups), lipid peroxidation (hydoxynonenal protein adducts), and muscular glutathione concentrations. HO expression was evaluated using anti-HO-1 and -HO-2 antibodies, and biological significance using HO inhibitor, and contractility studies. We conclude that the iNOS isoform plays a major role in nitrotyrosine formation and protein oxidation in septic ventilatory muscles, and that HO protect muscle contractile machinery from the deleterious effects of ROS in control and septic muscles.