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1.  Effects of heparin on the uptake of lipoprotein lipase in rat liver 
BMC Physiology  2004;4:13.
Lipoprotein lipase (LPL) is anchored at the vascular endothelium through interaction with heparan sulfate. It is not known how this enzyme is turned over but it has been suggested that it is slowly released into blood and then taken up and degraded in the liver. Heparin releases the enzyme into the circulating blood. Several lines of evidence indicate that this leads to accelerated flux of LPL to the liver and a temporary depletion of the enzyme in peripheral tissues.
Rat livers were found to contain substantial amounts of LPL, most of which was catalytically inactive. After injection of heparin, LPL mass in liver increased for at least an hour. LPL activity also increased, but not in proportion to mass, indicating that the lipase soon lost its activity after being bound/taken up in the liver. To further study the uptake, bovine LPL was labeled with 125I and injected. Already two min after injection about 33 % of the injected lipase was in the liver where it initially located along sinusoids. With time the immunostaining shifted to the hepatocytes, became granular and then faded, indicating internalization and degradation. When heparin was injected before the lipase, the initial immunostaining along sinusoids was weaker, whereas staining over Kupffer cells was enhanced. When the lipase was converted to inactive before injection, the fraction taken up in the liver increased and the lipase located mainly to the Kupffer cells.
This study shows that there are heparin-insensitive binding sites for LPL on both hepatocytes and Kupffer cells. The latter may be the same sites as those that mediate uptake of inactive LPL. The results support the hypothesis that turnover of endothelial LPL occurs in part by transport to and degradation in the liver, and that this transport is accelerated after injection of heparin.
PMCID: PMC534784  PMID: 15544705
2.  Seasonal ovulatory activity exists in tropical Creole female goats and Black Belly ewes subjected to a temperate photoperiod 
BMC Physiology  2004;4:12.
Seasonality of ovulatory activity is observed in European sheep and goat breeds, whereas tropical breeds show almost continuous ovulatory activity. It is not known if these tropical breeds are sensitive or not to temperate photoperiod. This study was therefore designed to determine whether tropical Creole goats and Black-Belly ewes are sensitive to temperate photoperiod. Two groups of adult females in each species, either progeny or directly born from imported embryos, were used and maintained in light-proof rooms under simulated temperate (8 to 16 h of light per day) or tropical (11 – 13 h) photoperiods. Ovulatory activity was determined by blood progesterone assays for more than two years. The experiment lasted 33 months in goats and 25 months in ewes.
Marked seasonality of ovulatory activity appeared in the temperate group of Creole female goats. The percentage of female goats experiencing at least one ovulation per month dramatically decreased from May to September for the three years (0%, 27% and 0%, respectively). Tropical female goats demonstrated much less seasonality, as the percentage of goats experiencing at least one ovulation per month never went below 56%. These differences were significant.
Both groups of temperate and tropical Black-Belly ewes experienced a marked seasonality in their ovulatory activity, with only a slightly significant difference between groups. The percentage of ewes experiencing at least one ovulation per month dropped dramatically in April and rose again in August (tropical ewes) or September (temperate ewes). The percentage of ewes experiencing at least one ovulation per month never went below 8% and 17% (for tropical and temperate ewes respectively) during the spring and summer months.
An important seasonality in ovulatory activity of tropical Creole goats was observed when females were exposed to a simulated temperate photoperiod. An unexpected finding was that Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the photoperiodic changes of the temperate zone even though they do not originate from these regions.
PMCID: PMC521071  PMID: 15333134
3.  Pattern of carbon dioxide production and retention is similar in adult pigs when fed hourly, but not when fed a single meal 
BMC Physiology  2004;4:11.
The understanding of bicarbonate kinetics and CO2 retention in the body is necessary to conduct amino acid tracer oxidation studies in both humans and laboratory animals. Significant metabolic activity is associated with eating which can affect bicarbonate steady state kinetics. A study was conducted to assess the impact of feeding regimen on the recovery of labelled bicarbonate and energy expenditure in adult female pigs (sows). Five catheterized sows (235 ± 5 kg) were fed semi-synthetic diets as: a single meal 2 h into the infusion after an overnight fast, or in eight hourly meals starting 2 h before the infusion. Oxygen consumption, CO2 production and 14CO2 recovery (ie fraction not retained) were determined during primed, constant intravenous infusions of NaH14CO3.
The 14CO2 recovery (%) after fasting (58.1 ± 4.8) was lower than that after single meal feeding (78.8 ± 5.9) or hourly meal feeding (81.0 ± 2.6, P = 0.03). CO2 production correlated with 14CO2 recovery during hourly feeding (r = 0.40, P = 0.01); this relationship was not significant after single meal feeding (P = 0.30), probably due to physical activity-associated CO2 production.
The correlation of CO2 retention factors with CO2 production during hourly feeding suggests that this regimen should be preferred for future amino acid kinetics studies.
PMCID: PMC476741  PMID: 15242516
4.  Differential expression of E-cadherin, N-cadherin and beta-catenin in proximal and distal segments of the rat nephron. 
BMC Physiology  2004;4:10.
The classical cadherins such as E- and N-cadherin are Ca2+-dependent cell adhesion molecules that play important roles in the development and maintenance of renal epithelial polarity. Recent studies have shown that a variety of cadherins are present in the kidney and are differentially expressed in various segments of the nephron. However, the interpretation of these findings has been complicated by the fact that the various studies focused on different panels of cadherins and utilized different species. Moreover, since only a few of the previous studies focused on the rat, information regarding the expression and localization of renal cadherins in this important species is lacking. In the present study, we have employed dual immunofluorescent labeling procedures that utilized specific antibodies against either E- or N-cadherin, along with antibodies that target markers for specific nephron segments, to characterize the patterns of cadherin expression in frozen sections of adult rat kidney.
The results showed that N-cadherin is the predominant cadherin in the proximal tubule, but is essentially absent in other nephron segments. By contrast, E-cadherin is abundant in the distal tubule, collecting duct and most medullary segments, but is present only at very low levels in the proximal tubule. Additional results revealed different patterns of N-cadherin labeling along various segments of the proximal tubule. The S1 and S2 segments exhibit a fine threadlike pattern of labeling at the apical cell surface, whereas the S3 segment show intense labeling at the lateral cell-cell contacts.
These results indicate that E- and N-cadherin are differentially expressed in the proximal and distal tubules of rat kidney and they raise the possibility that differences in cadherin expression and localization may contribute to the differences in the susceptibility of various nephron segments to renal pathology or nephrotoxic injury.
PMCID: PMC459230  PMID: 15147582
5.  Endurance training of respiratory muscles improves cycling performance in fit young cyclists 
BMC Physiology  2004;4:9.
Whether or not isolated endurance training of the respiratory muscles improves whole-body endurance exercise performance is controversial, with some studies reporting enhancements of 50 % or more, and others reporting no change. Twenty fit (VO2 max 56.0 ml/kg/min), experienced cyclists were randomly assigned to three groups. The experimental group (n = 10) trained their respiratory muscles via 20, 45 min sessions of hyperpnea. The placebo group (n = 4) underwent "sham" training (20, 5 min sessions), and the control group (n = 6) did no training.
After training, the experimental group increased their respiratory muscle endurance capacity by 12 %. Performance on a bicycle time trial test designed to last about 40 min improved by 4.7 % (9 of 10 subjects showed improvement). There were no test-re-test improvements in either respiratory muscle or bicycle exercise endurance performance in the placebo group, nor in the control group. After training, the experimental group had significantly higher ventilatory output and VO2, and lower PCO2, during constant work-rate exercise; the placebo and control groups did not show these changes. The perceived respiratory effort was unchanged in spite of the higher ventilation rate after training.
The results suggest that respiratory muscle endurance training improves cycling performance in fit, experienced cyclists. The relative hyperventilation with no change in respiratory effort sensations suggest that respiratory muscle training allows subjects to tolerate the higher exercise ventilatory response without more dyspnea. Whether or not this can explain the enhanced performance is unknown.
PMCID: PMC419707  PMID: 15132753
exercise; humans; oxygen consumption; pulmonary ventilation; time trial tests
6.  [HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells 
BMC Physiology  2004;4:8.
Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.
RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).
HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.
PMCID: PMC411047  PMID: 15117409
7.  Heart rate variability and short duration spaceflight: relationship to post-flight orthostatic intolerance 
BMC Physiology  2004;4:6.
Upon return from space many astronauts experience symptoms of orthostatic intolerance. Research has implicated altered autonomic cardiovascular regulation due to spaceflight with further evidence to suggest that there might be pre-flight autonomic indicators of post-flight orthostatic intolerance. We used heart rate variability (HRV) to determine whether autonomic regulation of the heart in astronauts who did or did not experience post-flight orthostatic intolerance was different pre-flight and/or was differentially affected by short duration (8 – 16 days) spaceflight. HRV data from ten-minute stand tests collected from the 29 astronauts 10 days pre-flight, on landing day and three days post-flight were analysed using coarse graining spectral analysis. From the total power (PTOT), the harmonic component was extracted and divided into high (PHI: >0.15 Hz) and low (PLO: = 0.15 Hz) frequency power regions. Given the distribution of autonomic nervous system activity with frequency at the sinus node, PHI/PTOT was used as an indicator of parasympathetic activity; PLO/PTOT as an indicator of sympathetic activity; and, PLO/PHI as an estimate of sympathovagal balance.
Twenty-one astronauts were classified as finishers, and eight as non-finishers, based on their ability to remain standing for 10 minutes on landing day. Pre-flight, non-finishers had a higher supine PHI/PTOT than finishers. Supine PHI/PTOT was the same pre-flight and on landing day in the finishers; whereas, in the non-finishers it was reduced. The ratio PLO/PHI was lower in non-finishers compared to finishers and was unaffected by spaceflight. Pre-flight, both finishers and non-finishers had similar supine values of PLO/PTOT, which increased from supine to stand. Following spaceflight, only the finishers had an increase in PLO/PTOT from supine to stand.
Both finishers and non-finishers had an increase in sympathetic activity with stand on pre-flight, yet only finishers retained this response on landing day. Non-finishers also had lower sympathovagal balance and higher pre-flight supine parasympathetic activity than finishers. These results suggest pre-flight autonomic status and post-flight impairment in autonomic control of the heart may contribute to orthostatic intolerance. The mechanism by which higher pre-flight parasympathetic activity might contribute to post-flight orthostatic intolerance is not understood and requires further investigation.
PMCID: PMC420472  PMID: 15113425
spectral analysis; orthostatic intolerance; stand test; parasympathetic nervous system; sympathetic nervous system
8.  Human cortical perfusion and the arterial pulse: a near-infrared spectroscopy study 
BMC Physiology  2004;4:7.
The pulsatile nature of the arterial pulse induces a pulsatile perfusion pattern which can be observed in human cerebral cortex with non-invasive near-infrared spectroscopy. The present study attempts to establish a quantitative relation between these two events, even in situations of very weak signal-to-noise ratio in the cortical perfusion signal. The arterial pulse pattern was extracted from the left middle finger by means of plethesmographic techniques. Changes in cortical perfusion were detected with a continuous-wave reflectance spectrophotometer on the scalp overlying the left prefrontal cortex. Cross-correlation analysis was performed to provide evidence for a causal relation between the arterial pulse and relative changes in cortical total hemoglobin. In addition, the determination of the statistical significance of this relation was established by the use of phase-randomized surrogates.
The results showed statistically significant cross correlation between the arterial and perfusion signals.
The approach designed in the present study can be utilized for a quantitative and continuous assessment of the perfusion states of the cerebral cortex in experimental and clinical settings even in situations of extremely low signal-to-noise ratio.
PMCID: PMC411046  PMID: 15113424
9.  Physiological studies in heterozygous calcium sensing receptor (CaSR) gene-ablated mice confirm that the CaSR regulates calcitonin release in vivo 
BMC Physiology  2004;4:5.
The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium.
To demonstrate regulatory effects of CaSR on calcitonin release, we studied calcitonin responsiveness to calcium in normal and CaSR heterozygous-ablated (Casr+/-) mice. Casr+/- mice have hypercalcemia and hypocalciuria, and live normal life spans. Each mouse received either 500 μl of normal saline or one of two doses of elemental calcium (500 μmol/kg or 5 mmol/kg) by intraperitoneal injection. Ionized calcium was measured at baseline and 10 minutes, and serum calcitonin was measured on the 10 minute sample.
At baseline, Casr+/- mice had a higher blood calcium, and in response to the two doses of elemental calcium, had greater increments and peak levels of ionized calcium than their wild type littermates. Despite significantly higher ionized calcium levels, the calcitonin levels of Casr+/- mice were consistently lower than wild type at any ionized calcium level, indicating that the dose-response curve of calcitonin to increases in ionized calcium had been significantly blunted or shifted to the right in Casr+/- mice.
These results confirm that the CaSR is a physiological regulator of calcitonin; therefore, in response to increases in ionized calcium, the CaSR inhibits parathyroid hormone secretion and stimulates calcitonin secretion.
PMCID: PMC419359  PMID: 15099400
Animal models/rodent; calcitonin; familial hypocalciuric hypocalcemia; calcium receptor; gene knock-out
10.  Detecting and minimizing zinc contamination in physiological solutions 
BMC Physiology  2004;4:4.
To explore the role of zinc (Zn) in cellular physiology it is important to be able to control and quantify the level of Zn contamination in experimental solutions. A technique that relies on a Zn-sensitive fluorimetric probe is introduced for measuring Zn concentrations as low as 100 pM. The method depends on the combination of the Zn-probe FluoZin-3 together with a slow Zn-chelator, Ca-EDTA, that reduces the background Zn levels and allows repeated measurements in the same solution.
The method was used to determine which common labware items could leach Zn into solution. Contamination was predictably found to arise from stainless steel and glass. Perhaps less expectedly it was also introduced by methacrylate cuvettes, plastic tissue culture dishes and other plastic labware. The release of nickel from stainless steel electrodes was also imaged using the fluorescent probe Newport Green.
Zn contamination may arise from rather unexpected sources; it is important that all aspects and components used in the course of an experiment be analyzed for the possibility of introducing contaminants.
PMCID: PMC395835  PMID: 15113426
11.  Some processes of energy saving and expenditure occurring during ethanol perfusion in the isolated liver of fed rats; a Nuclear Magnetic Resonance study. 
BMC Physiology  2004;4:3.
In the isolated liver of fed rats, a 10 mM ethanol perfusion rapidly induced a rapid 25% decrease in the total ATP content, the new steady state resulting from both synthesis and consumption. The in situ rate of mitochondrial ATP synthesis without activation of the respiration was increased by 27%, implying an increased energy demand. An attempt to identify the ethanol-induced ATP-consuming pathways was performed using 31P and 13C Nuclear Magnetic Resonance.
Ethanol (i) transiently increased sn-glycerol-3-phosphate formation whereas glycogenolysis was continuously maintained; (ii) decreased the glycolytic ATP supply and (iii) diminished the intracellular pH in a dose-dependent manner in a slight extend. Although the cytosolic oxidation of ethanol largely generated H+ (and NADH), intracellular pHi was maintained by (i) the large and passive excretion of cellular acetic acid arising from ethanol oxidation (evidenced by exogenous acetate administration), without energetic cost or (ii) proton extrusion via the Na+-HCO3- symport (implying the indirect activation of the Na+-K+-ATPase pump and thus an energy use), demonstrated during the addition of their specific inhibitors SITS and ouabaïn, respectively.
Various cellular mechanisms diminish the cytosolic concentration of H+ and NADH produced by ethanol oxidation, such as (i) the large but transient contribution of the dihydroxyacetone phosphate / sn-glycerol-3-phosphate shuttle between cytosol and mitochondria, mainly implicated in the redox state and (ii) the major participation of acetic acid in passive proton extrusion out of the cell. These processes are not ATP-consuming and the latter is a cellular way to save some energy. Their starting in conjunction with the increase in mitochondrial ATP synthesis in ethanol-perfused whole liver was however insufficient to alleviate either the inhibition of glycolytic ATP synthesis and/or the implication of Na+-HCO3- symport and Na+-K+-ATPase in the pHi homeostasis, energy-consuming carriers.
PMCID: PMC375537  PMID: 15053831
12.  Exercise increases endostatin in circulation of healthy volunteers 
BMC Physiology  2004;4:2.
Physical inactivity increases the risk of atherosclerosis. However, the molecular mechanisms of this relation are poorly understood. A recent report indicates that endostatin, an endogenous angiostatic factor, inhibits the progression of atherosclerosis, and suggests that reducing intimal and atherosclerotic plaque tissue neovascularization can inhibit the progression atherosclerosis in animal models. We hypothesize that exercise can elevate the circulatory endostatin level. Hence, exercise can protect against one of the mechanisms of atherosclerosis.
We examined treadmill exercise tests in healthy volunteers to determine the effect of exercise on plasma levels of endostatin and other angiogenic regulators. Oxygen consumption (VO2) was calculated. Plasma levels of endostatin, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) were determined using ELISA. The total peak VO2 (L) in 7 male subjects was 29.5 ± 17.8 over a 4–10 minute interval of exercise. Basal plasma levels of endostatin (immediately before exercise) were 20.3 ± 3.2 pg/ml, the plasma levels increased to 29.3 ± 4.2, 35.2 ± 1.8, and 27.1 ± 2.2 ng/ml, at 0.5, 2, and 6 h, respectively, after exercise. There was a strong linear correlation between increased plasma levels of endostatin (%) and the total peak VO2 (L) related to exercise (R2 = 0.9388; P < 0.01). Concurrently, VEGF levels decreased to 28.3 ± 6.4, 17.6 ± 2.4, and 26.5 ± 12.5 pg/ml, at 0.5, 2, and 6 h, respectively, after exercise. There were no significant changes in plasma bFGF levels in those subjects before and after exercise.
The results suggest that circulating endostatin can be significantly increased by exercise in proportion to the peak oxygen consumption under physiological conditions in healthy volunteers. These findings may provide new insights into the molecular links between physical inactivity and the risk of angiogenesis dependent diseases such as atherosclerosis.
PMCID: PMC324413  PMID: 14728720
13.  Heterotrimeric G protein subunits are located on rat liver endosomes 
BMC Physiology  2004;4:1.
Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors.
By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM) and basolateral (BLM) membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC) > compartment for uncoupling of receptor and ligand (CURL) > multivesicular bodies (MVB) >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC) was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers.
We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s) of internalized insulin receptors.
PMCID: PMC324412  PMID: 14711382

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