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1.  Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments 
BMC Physiology  2001;1:15.
Background
The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments.
Results
Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S) expressed the N-terminal half of α1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current.
Discussion
A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.
PMCID: PMC64647  PMID: 11806762
2.  Age-dependent changes in 24-hour rhythms of catecholamine content and turnover in hypothalamus, corpus striatum and pituitary gland of rats injected with Freund's adjuvant 
BMC Physiology  2001;1:14.
Background
Little information is available on the circadian sequela of an immune challenge in the brain of aged rats. To assess them, we studied 24-hour rhythms in hypothalamic and striatal norepinephrine (NE) content, hypothalamic and striatal dopamine (DA) turnover and hypophysial NE and DA content, in young (2 months) and aged (18–20 months) rats killed at 6 different time intervals, on day 18th after Freund's adjuvant or adjuvant's vehicle administration.
Results
Aging decreased anterior and medial hypothalamic NE content, medial and posterior hypothalamic DA turnover, and striatal NE concentration and DA turnover. Aging also decreased NE and DA content in pituitary neurointermediate lobe and augmented DA content in the anterior pituitary lobe. Immunization by Freund's adjuvant injection caused: (i) reduction of DA turnover in anterior hypothalamus and corpus striatum; (ii) acrophase delay of medial hypothalamic DA turnover in old rats, and of striatal NE content in young rats; (iii) abolition of 24-h rhythm in NE and DA content of neurointermediate pituitary lobe, and in DA content of anterior lobe, of old rats.
Conclusions
The decline in catecholamine neurotransmission with aging could contribute to the decrease of gonadotropin and increase of prolactin release reported in similar groups of rats. Some circadian responses to immunization, e.g. suppression of 24-h rhythms of neurointermediate lobe NE and DA and of anterior lobe DA were seen only in aged rats.
PMCID: PMC61031  PMID: 11741510
3.  The dynamics of venous return and response to hypervolemia in the toad, Bufo marinus (L.) 
BMC Physiology  2001;1:13.
Background
Venous return from the posterior region of amphibians travels by either two renal portal veins to the kidney or a central abdominal vein that drains into the hepatic portal system. The relative proportions of blood flow in these vessels has never been measured nor has a modification of flow been determined when venous return increases by changes in blood volume during hypervolemia or during increased volume input from the posterior lymph hearts.
Results
Venous return from the posterior region of Bufo marinus was measured under resting conditions and in response to a systemic hypervolemia. Doppler flow probes were positioned on the renal portal and ventral abdominal veins, and flow was recorded as injections of artificial plasma equaling 100% of the animal's plasma volume were administered through the sciatic artery. Resting flow was found to be 5.54 ± 2.03 ml min-1 kg-1 in the paired renal portal veins, and 7.31 ± 0.89 ml min-1 kg-1 in the ventral abdominal vein. While renal portal flow was found to increase by a factor of 2.4 times during the first 10 min of hypervolemia, ventral abdominal flow only increased by a factor of 1.3.
Conclusions
Our results quantify the contribution to circulation from posterior venous return in the toad Bufo marinus. A preferential movement of excess fluid through the renal portal pathway was also demonstrated, supporting the possibility of water elimination via the renal portal circulation, especially during periods of high water influx into the animals.
PMCID: PMC59660  PMID: 11696249
4.  High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition 
BMC Physiology  2001;1:12.
Background
In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the β-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation.
Results
Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 ± 10 vs 63 ± 10 mmHg, p < 0.05), though peak systolic intracellular calcium was not significantly different, suggesting reduced myofilament responsiveness to intracellular calcium with high perfusate calcium. The ratio of developed pressure to myocardial oxygen consumption, an index of economy of contraction, was significantly increased with dobutamine compared to high perfusate calcium (1.35 ± 0.15 vs 1.15 ± 0.15 mmHg/μmoles/min/g dry wt, p < 0.05), suggesting energetic inefficiency with high perfusate calcium. The specific protein kinase C inhibitor, chelerythrine, significantly attenuated the expected increase in developed pressure when increasing perfusate calcium from 2.5 to 3.5 mM (3.5 mM: 64 ± 8 vs 3.5 mM + chelerythrine: 55 ± 5 mmHg, p < 0.05), though had no effects on dobutamine, or lower levels of perfusate calcium (1.5 to 2.5 mM).
Conclusions
By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation.
PMCID: PMC55339  PMID: 11553322
5.  1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line 
BMC Physiology  2001;1:11.
Background
The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.
Results
In the current study, we have demonstrated that Caco-2 cells express low but detectable levels of CaT1 mRNA in the absence of 1,25-dihydroxyvitamin D treatment. CaT1 mRNA expression is rapidly up regulated (4-fold increase at 4 h and 10-fold at 24 h) by treatment with 1,25-dihydroxyvitamin D (10-7 moles/L). Moreover, the increase in CaT1 mRNA expression preceded by several hours the vitamin D induction of calbindin D9K, a putative cytosolic calcium transport protein.
Conclusion
These observations are the first to demonstrate regulation of CaT1 expression by vitamin D and are consistent with a new model of intestinal calcium absorption wherein vitamin D-mediated changes in brush border membrane CaT1 levels could be the primary gatekeeper regulating homeostatic modulation of intestinal calcium absorption efficiency.
PMCID: PMC55338  PMID: 11545681
6.  Localization of heat shock protein 20 in swine carotid artery 
BMC Physiology  2001;1:10.
Background
Cyclic nucleotides can relax vascular smooth muscle by mechanisms distal to myosin regulatory light chain (MRLC) phosphorylation. This mechanism, termed relaxation without MRLC dephosphorylation, may be regulated by ser16 phosphorylation of heat shock protein 20 (HSP20).
Results
Confocal imaging of HSP20 in smooth muscle tissues revealed that HSP20 was present throughout the cytoplasm, although some focal regions of the cytoplasm were found to contain more HSP20 than the remaining cytoplasm. The distribution of HSP20 within the cytoplasm was not altered by histamine, forskolin, or nitroglycerin.
Conclusion
Cytoplasmic localization of HSP20 is consistent with a potential function of HSP20 as a regulator of smooth muscle contractile force.
PMCID: PMC48151  PMID: 11532202
7.  Dopaminergic neurons in the brain and dopaminergic innervation of the albumen gland in mated and virgin helisoma duryi (mollusca: pulmonata) 
BMC Physiology  2001;1:9.
Background
Dopamine was shown to stimulate the perivitelline fluid secretion by the albumen gland. Even though the albumen gland has been shown to contain catecholaminergic fibers and its innervation has been studied, the type of catecholamines, distribution of fibers and the precise source of this neural innervation has not yet been deduced. This study was designed to address these issues and examine the correlation between dopamine concentration and the sexual status of snails.
Results
Dopaminergic neurons were found in all ganglia except the pleural and right parietal, and their axons in all ganglia and major nerves of the brain. In the albumen gland dopaminergic axons formed a nerve tract in the central region, and a uniform net in other areas. Neuronal cell bodies were present in the vicinity of the axons. Dopamine was a major catecholamine in the brain and the albumen gland. No significant difference in dopamine quantity was found when the brain and the albumen gland of randomly mating, virgin and first time mated snails were compared.
Conclusions
Our results represent the first detailed studies regarding the catecholamine innervation and quantitation of neurotransmitters in the albumen gland. In this study we localized catecholaminergic neurons and axons in the albumen gland and the brain, identified these neurons and axons as dopaminergic, reported monoamines present in the albumen gland and the brain, and compared the dopamine content in the brain and the albumen gland of randomly mating, virgin and first time mated snails.
PMCID: PMC37538  PMID: 11513757
8.  Modulation of L-type Ca2+ current but not activation of Ca2+ release by the gamma1 subunit of the dihydropyridine receptor of skeletal muscle 
BMC Physiology  2001;1:8.
Background
The multisubunit (α1S,α2-δ, β1a and γ1) skeletal muscle dihydropyridine receptor (DHPR) transduces membrane depolarization into release of Ca2+ from the sarcoplasmic reticulum (SR) and also acts as an L-type Ca2+ channel. To more fully investigate the function of the γ1 subunit in these two processes, we produced mice lacking this subunit by gene targeting.
Results
Mice lacking the DHPR γ1 subunit (γ1 null) survive to adulthood, are fertile and have no obvious gross phenotypic abnormalities. The γ1 subunit is expressed at approximately half the normal level in heterozygous mice (γ1 het). The density of the L-type Ca2+ current in γ1 null and γ1 het myotubes was higher than in controls. Inactivation of the Ca2+ current produced by a long depolarization was slower and incomplete in γ1 null and γ1 het myotubes, and was shifted to a more positive potential than in controls. However, the half-activation potential of intramembrane charge movements was not shifted, and the maximum density of the total charge was unchanged. Also, no shift was observed in the voltage-dependence of Ca2+ transients. γ1 null and γ1 het myotubes had the same peak Ca2+ amplitude vs. voltage relationship as control myotubes.
Conclusions
The L-type Ca2+ channel function, but not the SR Ca2+ release triggering function of the skeletal muscle dihydropyridine receptor, is modulated by the γ1 subunit.
PMCID: PMC37314  PMID: 11495636
9.  Pathological apoptosis by xanthurenic acid, a tryptophan metabolite: activation of cell caspases but not cytoskeleton breakdown 
BMC Physiology  2001;1:7.
Background
A family of aspartate-specific cysteinyl proteases, named caspases, mediates programmed cell death, apoptosis. In this function, caspases are important for physiological processes such as development and maintenance of organ homeostasis. Caspases are, however, also engaged in aging and disease development. The factors inducing age-related caspase activation are not known. Xanthurenic acid, a product of tryptophan degradation, is present in blood and urine, and accumulates in organs with aging.
Results
Here, we report triggering of apoptotic key events by xanthurenic acid in vascular smooth muscle and retinal pigment epithelium cells. Upon exposure of these cells to xanthurenic acid a degradation of ICAD/DFF45, poly(ADP-ribose) polymerase, and gelsolin was observed, giving a pattern of protein cleavage characteristic for caspase-3 activity. Active caspase-3, -8 and caspase-9 were detected by Western blot analysis and immunofluorescence. In the presence of xanthurenic acid the amino-terminal fragment of gelsolin bound to the cytoskeleton, but did not lead to the usually observed cytoskeleton breakdown. Xanthurenic acid also caused mitochondrial migration, cytochrome C release, and destruction of mitochondria and nuclei.
Conclusions
These results indicate that xanthurenic acid is a previously not recognized endogenous cell death factor. Its accumulation in cells may lead to accelerated caspase activation related to aging and disease development.
PMCID: PMC34606  PMID: 11459518
10.  Method for non-invasively recording electrocardiograms in conscious mice 
BMC Physiology  2001;1:6.
Background
The rapid increase in the development of mouse models is resulting in a growing demand for non-invasive physiological monitoring of large quantities of mice. Accordingly, we developed a new system for recording electrocardiograms (ECGs) in conscious mice without anesthesia or implants, and created Internet-accessible software for analyzing murine ECG signals. The system includes paw-sized conductive electrodes embedded in a platform configured to record ECGs when 3 single electrodes contact 3 paws.
Results
With this technique we demonstrated significantly reduced heart rate variability in neonates compared to adult mice. We also demonstrated that female mice exhibit significant ECG differences in comparison to age-matched males, both at baseline and in response to β-adrenergic stimulation.
Conclusions
The technology we developed enables non-invasive screening of large numbers of mice for ECG changes resulting from genetic, pharmacological, or pathophysiological alterations. Data we obtained non-invasively are not only consistent with what have been reported using invasive and expensive methods, but also demonstrate new findings regarding gender-dependent and age-dependent variations in ECGs in mice.
PMCID: PMC35354  PMID: 11476671
11.  Endothelial [Ca2+]i is an integrating signal for the vascular tone in rat aortae 
BMC Physiology  2001;1:5.
Background
Although various endothelium-dependent relaxing factors (endothelial autacoids) are released upon the elevation of endothelial cytosolic free Ca2+ concentration (EC [Ca2+]i), the quantitative relationship between EC [Ca2+]i and vascular tone remains to be established. Moreover, whether the basal release of endothelial autacoids is modulated by basal EC [Ca2+]i is still unclear. We assessed these issues by using a novel method that allows simultaneous recording of EC [Ca2+]i and vascular displacement in dissected rat aortic segments.
Results
Receptor-dependent (acetylcholine) or independent (ionomycin) agonists caused immediate EC [Ca2+]i elevation followed by vasorelaxation in preparations pre-contracted with phenylephrine. Low doses of agonists induced small EC [Ca2+]i elevations (about 100 nmol/L) and concomitant half-maximal vasorelaxation. At high doses, agonists elevated EC [Ca2+]i to μmol/L range with little additional vasodilatation. When EC [Ca2+]i was plotted against the vasorelaxation, the curves were almost identical for both acetylcholine and ionomycin treatments, in the presence or absence of various endothelial autacoid inhibitors. Calcium-free solution reduced basal EC [Ca2+]i and induced a drastic vasoconstriction. Endothelial autacoid inhibitors reduced EC [Ca2+]i changes and abolished both agonist-induced vasodilatation and calcium-free solution-induced vessel contraction. When the EC [Ca2+]i was completely chelated by 40 μmol/L BAPTA, the acetylcholine-evoked vasorelaxation could be abolished as well. However, when the EC [Ca2+]i was partially chelated by 20 μmol/L BAPTA, the acetylcholine-evoked vasorelaxation was almost unaffected.
Conclusions
These results indicate that vascular tone is modulated by subtle changes of EC [Ca2+]i level, which seems to serve as an integrating signal in both basal and stimulated states.
PMCID: PMC34108  PMID: 11434869
12.  Calcium-mediated transductive systems and functionally active gap junctions in astrocyte-like GL15 cells 
BMC Physiology  2001;1:4.
Background
It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein.
Results
The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation.
Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl).
Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes.
Conclusions
In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.
doi:10.1186/1472-6793-1-4
PMCID: PMC32183  PMID: 11384510
13.  Functional interaction between TRP4 and CFTR in mouse aorta endothelial cells 
BMC Physiology  2001;1:3.
Background
This study describes the functional interaction between the putative Ca2+ channel TRP4 and the cystic fibrosis transmembrane conductance regulator, CFTR, in mouse aorta endothelium (MAEC).
Results
MAEC cells express CFTR transcripts as shown by RT-PCR analysis. Application of a phosphorylating cocktail activated a Cl- current with characteristics similar to those of CFTR mediated currents in other cells types (slow activation by cAMP, absence of rectification, block by glibenclamide). The current is present in trp4 +/+ MAEC, but not in trp4 -/- cells, although the expression of CFTR seems unchanged in the trp4 deficient cells as judged from RT-PCR analysis.
Conclusions
It is concluded that TRP4 is necessary for CFTR activation in endothelium, possibly by providing a scaffold for the formation of functional CFTR channels.
PMCID: PMC32182  PMID: 11356184
14.  Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells 
BMC Physiology  2001;1:2.
Background
Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells.
Results
Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation.
Conclusions
These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.
doi:10.1186/1472-6793-1-2
PMCID: PMC32181  PMID: 11231888
15.  Differential expression of Aquaporin 8 in human colonic epithelial cells and colorectal tumors 
BMC Physiology  2001;1:1.
Background
The gene expression pattern in tumor cells differs from that in corresponding normal cells. In order to identify differentially expressed genes in colorectal tumors and normal colorectal epithelium, a differential display experiment was used to compare RNA expression in normal and tumor tissue samples.
Results
One gene fragment was expressed only in normal tissue and not, or to a much lesser extent, in the adenomas, carcinomas and cancer cell lines. The isolated gene fragment was identical to Aquaporin 8 (AQP8), a water channel protein. In situ hybridization demonstrated that AQP8 was expressed in the cells facing the lumen in the normal colonic epithelium.
Conclusion
Our result suggests that the expression of AQP8 is a marker of normal proliferating colonic epithelial cells and suggest these cells to be involved in fluid transport in the colon.
PMCID: PMC32180  PMID: 11231887

Results 1-15 (15)