bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanical Stress‐induced DNA damage and rac‐p38MAPK Signal Pathways Mediate p53‐dependent Apoptosis in Vascular Smooth Muscle Cells
Authors: Mayr Manuel , Hu Yanhua , Hainaut Pierre , Xu Qingbo
Published: August 2002

Recently, we demonstrated that biomechanical stress induces apoptosis of vascular smooth muscle cells (SMCs) (Mayr et al., FASEB J. 2000; 15:261.270). In this article we investigated the molecular mechanisms of mechanical stress‐induced apoptosis. When SMCs were subjected to cyclic strain, tumor‐suppressor p53 was activated as evidenced by gel mobility shift assays and Western blot analyses. p53 activation was largely attenuated if SMCs were pretreated with SB202190, a specific p38MAPK inhibitor, or were stably transfected with dominant negative rac, an upstream signal transducer of p38MAPK pathways. Kinase assays provided direct evidence that p38MAPKs phosphorylated p53 within 30 min of cyclic strain. Additionally, mechanical stress resulted in oxidative DNA damage as detected by the presence of 8‐oxoguanine. Treatment with the antioxidant U‐74389G abrogated p53 activation. p53 activation was followed by expression and mitochondrial translocation of the proapoptotic protein Bax. Likewise, mechanical stress resulted in up‐regulation of anti‐apoptotic Bcl‐2 proteins, including Bcl‐2 and Bcl‐xL. However, a marked loss of mitochondrial membrane potential occurred in wild‐type, but not in p53−/−, SMCs. The latter lost their ability to express Bax and showed no apoptosis in response to cyclic strain. Taken together, our data provide the first evidence that SMC apoptosis induced by mechanical stress is p53‐dependent.
Glycosulfopeptides modeled on P‐selectin glycoprotein ligand‐1 inhibit P‐selectin‐dependent leukocyte rolling in vivo
Authors: Hicks Anne E.R. , Leppänen Anne , Cummings Richard D. , McEver Rodger P. , Hellewell Paul G. , Norman Keith E.
Published: August 2002

Leukocytic inflammation can be limited by inhibiting selectin‐dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure‐activity profiles, inhibition of P‐selectin‐dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL‐1, inhibit leukocyte binding to P‐selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P‐selectin‐dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P‐selectin‐dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2‐GSP‐6 and 4‐GSP‐6 substantially reversed P‐selectindependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2‐ and 4‐GSP‐6 lasted 2–4 min. Clearance studies with 125Ilabeled 4‐GSP‐6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P‐selectin ligand can inhibit P‐selectin‐dependent leukocyte rolling in vivo.
Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2dependent mechanism
Authors: Tithof Patricia K. , Elgayyar Mona , Cho Yeesook , Guan Wei , Fisher Aron B. , Peters‐Golden Marc
Published: August 2002

Smoking is a major risk factor for endothelial cell injury and subsequent coronary artery disease. Epidemiological studies implicate the phospholipase A2/arachidonic acid cascade in the mechanism by which smoking causes heart disease. However, specific components of cigarette smoke that activate this pathway have not been identified. The purpose of this study was to investigate the effects of polycyclic aromatic hydrocarbons contained in cigarette smoke on phospholipase A2 (PLA2) activity and apoptosis of human coronary artery endothelial cells. 1methylanthracene (1‐MA), phenanthrene (PA), and benzo(a)pyrene (B(a)P) caused significant release of 3H‐arachidonate from endothelial cells. 1‐MA and PA, but not B(a)P, also caused significant release of 3H‐linoleic acid. Release of fatty acids from membrane phospholipids preceded the onset of apoptosis. 3H‐arachidonate release and apoptosis induced by 1‐MA, B(a)P, and PA were inhibited by methylarachidonoyl‐fluorophosphonate, an inhibitor of Groups IV and VI PLA2s. Bromoenol lactone, an inhibitor of Group VI enzymes, inhibited both 3H‐arachidonate release and apoptosis induced by 1‐MA and PA, but not B(a)P. MJ33, an inhibitor of the acidic calcium‐independent PLA2, attenuated 3H‐arachidonate release and apoptosis by PA, but not 1MA or B(a)P. The presence of Groups IV and VI and the acidic iPLA2 in endothelial cells was demonstrated by reverse transcriptase‐polymerase chain reaction and Western analysis. These data suggest that 1‐MA, B(a)P and PA induce apoptosis of endothelial cells by a mechanism that involves activation of these three distinct isoforms of PLA2.
Innervation‐dependent phosphorylation and accumulation of αB–crystallin and Hsp27 as insoluble complexes in disused muscle
Authors: Kato Kanefusa , Ito Hidenori , Kamei Keiko , Iwamoto Ikuko , Inaguma Yutaka
Published: August 2002

Levels and phosphorylation states of the two small molecular chaperones, αB‐crystallin and Hsp27, in disused rat soleus muscles were determined by Western blot analysis of extracts with antibodies recognizing each of the two proteins and their phosphorylated serine residues. Increased phosphorylation and relocalization to insoluble fractions were found within a few days of hind‐limb suspension. High phosphorylation of αB‐crystallin at Ser‐59 (and to a certain extent, at Ser‐45) and of Hsp27 at Ser‐15 and Ser‐85, along with phosphorylated, active states of p38 and p44/42 mitogen‐activated protein kinases were maintained during hind‐limb suspension but promptly returned to control levels within a 5‐day recovery period. These results are similar to those observed with U373 MG glioma cells exposed to proteasome inhibitors (16). However, the responses of αB‐crystallin and Hsp27 in suspended soleus muscles did not appear with ipsilateral transection of the sciatic nerve trunk, indicating mediation by nerve activity. The fact that ubiquitinated proteins accumulated in the insoluble fractions of suspended soleus muscle further suggests participation of αB‐crystallin and Hsp27 in quality control of proteins in disused soleus muscle, with involvement of nerve activity‐dependent processes.
The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors, which increase PSA secretion and modify actin cytoskeleton
Authors: Kampa M. , Papakonstanti E.A. , Hatzoglou A. , Stathopoulos E.N. , Stournaras C. , Castanas E.
Published: August 2002

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short‐time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non‐internalizable analog testosterone‐BSA increase rapidly the release of prostate‐specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non‐internalizable fluorescent analog testosterone‐BSA‐FITC, on whole cells. Binding on these sites is time‐ and concentration‐dependent and specific for testosterone, presenting a Kd of 10.9 nM and a number of 144 sites/mg protein (~13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.
Interleukin‐1α (IL‐1α) promotes angiogenesis in vivo via VEGFR‐2 pathway by inducing inflammatory cell VEGF synthesis and secretion
Authors: Salven Petri , Hattori Koichi , Heissig Beate , Rafii Shahin
Published: August 2002

During inflammation, functional changes occur in the vasculature, including extensive endothelial cell mitotic activity and remodeling of capillaries. Interleukin‐1α. (IL‐1α) is a prototypical proinflammatory cytokine. Vascular endothelial growth factor (VEGF) is a strong endothelial cell‐specific mitogen that exerts a pivotal role in angiogenesis under physiological and pathological conditions. We show that IL‐1α stimulates VEGF secretion by human peripheral blood mononuclear cells (PBMNCs) in a dose‐dependent manner. This represents induction of de novo VEGF synthesis, as an induction of VEGF mRNA was observed. Also, the release of VEGF was blocked by cycloheximide. Reverse transcription‐polymerase chain reaction (RT‐PCR) detected four VEGF splice variants in unstimulated and in IL‐1α‐stimulated PBMNCs. in vivo in mice, subcutaneously administered IL‐1α caused a strong local angiogenic response, which was accompanied by an infiltrate of VEGF‐expressing inflammatory cells. The angiogenic effect of IL‐1α was blocked when the mice were treated with VEGF receptor 2 (VEGFR‐2) neutralizing antibodies. VEGFR‐1 blocking antibodies had a marginal inhibitory effect on IL‐1α‐induced angiogenesis. These observations indicate that IL‐1α induces angiogenesis by activating the VEGF‐VEGFR‐2 signaling pathway between inflammatory cells and blood vessel endothelial cells. This novel mechanism of IL‐1α‐action may enhance the shift to angiogenic phenotype in various conditions designated by excessive angiogenesis.
Protein kinase C‐ε mediates bradykinin‐induced cyclooxygenase‐2 expression in human airway smooth muscle cells
Authors: Pang Linhua , Nie Mei , Corbett Lisa , Donnelly Richard , Gray Samuel , Knox Alan J.
Published: August 2002

We previously reported that proinflammatory mediator bradykinin (BK) induces cyclooxygenase (COX)‐2 expression in human airway smooth muscle (HASM), but the mechanism is unknown in any biological system. Here, we studied the role of specific protein kinase C (PKC) isozyme(s) in COX‐2 expression. Among the eight PKC isozymes present in HASM cells, the Ca2+‐independent PKC‐δ and ‐ε and the Ca2+‐dependent PKC‐α and ‐βI were translocated to the nucleus upon BK stimulation. BK‐induced COX‐2 expression and prostaglandin E2 (PGE2) accumulation were mimicked by the direct PKC activator phorbol 12‐myristate 13‐acetate (PMA) and inhibited by the broad spectrum PKC inhibitor bisindolylmaleimide I. However, the selective Ca2+‐dependent PKC isozyme inhibitor Go 6976 had no effect. Furthermore, the membrane‐permeable calcium chelator BAPTA‐AM had no effect on BK‐induced COX‐2 expression and COX activity despite its inhibition of PGE2 accumulation, suggesting the involvement of Ca2+‐independent PKC isozymes. Rottlerin, a PKC‐δ inhibitor, also had no effect, likely implicating PKC‐ε. BK‐stimulated transcriptional activation of a COX‐2 promoter reporter construct was enhanced by overexpression of wild‐type PKC‐ε and abolished by a dominant negative PKC‐ε, but it was not affected by wild‐type or dominant negative PKC‐α or ‐δ. Collectively, our results demonstrate that PKC‐ε mediates BK‐induced COX‐2 expression in HASM cells.
Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of KATP channel opening
Authors: Patel Hemal H. , Ludwig Lynda M. , Fryer Ryan M. , Hsu Anna K. , Warltier David C. , Gross Garrett J.
Published: August 2002

Opioids and volatile anesthetics produce marked cardioprotective effects against myocardial infarction via the activation of ATP‐sensitive potassium (KATP) channels, however, the effect of combined treatment with both drugs is unknown. We examined the hypothesis that opioids and volatile anesthetics potentiate cardiac KATP channel opening, thereby enhancing cardioprotection. Rats were treated with the delta opioid agonists, TAN‐67 or BW373U86, or isoflurane, together or alone with and without diazoxide, a mitochondrial KATP channel opener. Glibenclamide, a non‐selective KATP channel blocker, was used to further characterize the signaling mechanism involved. Myocardial infarct size (IS) was determined by tetrazolium staining and was expressed as a percent of the area at risk (AAR). High doses of TAN‐67 (10 mg/kg), diazoxide (10 mg/kg), and isoflurane (1 MAC) produced a significant reduction in IS compared with the control group (30±3%, 36±5%, and 42±2 vs. 58±2%, respectively), whereas lower doses of the drugs had no effect except for the low dose of isoflurane (0.5 MAC). The combination of TAN‐67 and diazoxide or isoflurane and diazoxide resulted in a marked reduction in IS compared with controls in the presence of high (9±3% and 14±3%) and low (17±7% and 31±7%) dose combinations, respectively. The combination of TAN‐67 or BW373U86 and isoflurane also caused a striking reduction in IS/AAR (16±7% and 7±2%, respectively). To date, this is the first demonstration that opioids and volatile anesthetics work in conjunction to confer protection against myocardial infarction through potentiation of cardiac KATP channel opening.
Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity
Authors: Williamson Angela L. , Brindley Paul J. , Abbenante Giovanni , Prociv Paul , Berry Colin , Girdwood Karen , Pritchard David I. , Fairlie David P. , Hotez Peter J. , Dalton John P. , Loukas Alex
Published: August 2002

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host‐parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D‐like protease from the canine hookworm Ancylosotoma caninum (Ac‐APR‐1) and the orthologous protease from the human hookworm Necator americanus (Na‐APR‐1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host‐parasite compatibility and host species range.
Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: Implications for Parkinson's disease
Authors: Sriram Krishnan , Matheson Joanna M. , Benkovic Stanley A. , Miller Diane B. , Luster Michael I. , O'Callaghan James P.
Published: August 2002

The pathogenic mechanisms underlying idiopathic Parkinson's disease (PD) remain enigmatic. Recent findings suggest that inflammatory processes are associated with several neurodegenerative disorders, including PD. Enhanced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)‐α, has been found in association with glial cells in the substantia nigra of patients with PD. To determine the potential role for TNF‐α in PD, we examined the effects of the 1‐methyl‐4‐phenyl‐1,2,3,4‐tetrahydropyridine (MPTP), a dopaminergic neurotoxin that mimics some of the key features associated with PD, using transgenic mice lacking TNF receptors. Administration of MPTP to wild‐type (+/+) mice resulted in a time‐dependent expression of TNF‐α in striatum, which preceded the loss of dopaminergic markers and reactive gliosis. In contrast, transgenic mice carrying homozygous mutant alleles for both the TNF receptors (TNFR‐DKO), but not the individual receptors, were completely protected against the dopaminergic neurotoxicity of MPTP. The data indicate that the proinflammatory cytokine TNF‐α is an obligatory component of dopaminergic neurodegeneration. Moreover, because TNF‐α is synthesized predominantly by microglia and astrocytes, our findings implicate the participation of glial cells in MPTP‐induced neurotoxicity. Similar mechanisms may underlie the etiopathogenesis of PD.
Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25‐dihydroxyvitamin D3 in 25hydroxyvitamin D3‐1α‐hydroxylase knockout mice
Authors: HOENDEROP JOOST G. J. , DARDENNE OLIVIER , VAN ABEL MONIQUE , VAN DER KEMP ANNEMIETE W. C. M. , VAN OS CAREL H. , ST.‐ARNAUD RENÉ , BINDELS RENEJ. M.
Published: August 2002

Pseudovitamin D‐deficiency rickets (PDDR) is an autosomal disease characterized by hyperparathyroidism, rickets, and undetectable levels of 1,25‐dihydroxyvitaminD3 (1,25(OH)2D3). Mice in which the 25hydroxyvitamin D3‐1α‐hydroxylase (1α‐OHase) gene was inactivated presented the same clinical phenotype as patients with PDDR and were used to study renal expression of the epithelial Ca2+ channel (ECaC1), the calbindins, Na+/Ca2+ exchanger (NCX1), and Ca2+‐ATPase (PMCA1b). Serum Ca2+ (1.20±0.05 mM) and mRNA/ protein expression of ECaC1 (41±3%), calbindin‐D28K (31±2%), calbindin‐D9K (58±7%), NCX1 (10±2%), PMCA1b (96±4%) were decreased in 1α‐OHase−/−mice compared with 1α‐OHase+/‐littermates. Feeding these miceaCa2+‐enriched diet normalized serum Ca2+ levels and expression of Ca2+ proteins except for calbindin‐D9K expression. 1,25(OH)2D3 repletion resulted in increased expression of Ca2+ transport proteins and normalization of serum Ca2+ levels. Localization of Ca2+ transport proteins was clearly polarized in which ECaC1 was localized along the apical membrane, calbindin‐D28K in the cytoplasm, and calbindin‐D9K along the apical and basolateral membranes, resulting in a comprehensive mechanism facilitating renal transcellular Ca2+ transport. This study demonstrated that high dietary Ca2+ intake is an important regulator of the renal Ca2+ transport proteins in 1,25(OH)2D3‐deficient status and thus contributes to the normalization of blood Ca2+ levels.—Hoenderop, J. G. J., Dardenne, O., van Abel, M., van der Kemp, A. W. C. M., van Os, C. H., St.—Arnaud, R., Bindels, R. J. M. Modulation of renal Ca2+ transport protein genes by dietary Ca2+ and 1,25‐dihydroxyvitamin D3 in 25 hydroxyvitamin D3‐1α‐hydroxylase knockout mice. FASEB J. 16, 1398–1406 (2002)
The fetal origins of atherosclerosis: maternal hypercholesterolemia, and cholesterol‐lowering or antioxidant treatment during pregnancy influence in utero programming and postnatal susceptibility to atherogenesis
Authors: PALINSKI WULF , NAPOLI CLAUDIO
Published: August 2002

It has long been postulated that pathogenic events during fetal development influence atherosclerosis‐related diseases later in life, but the mechanisms involved are unknown. This review focuses on the evidence indicating that maternal hypercholesterolemia during pregnancy is responsible for one cascade of pathogenic events. Maternal hypercholesterolemia is associated with greatly increased fatty streak formation in human fetal arteries and accelerated progression of atherosclerosis during childhood. Recent experiments in genetically more homogeneous rabbits established that temporary diet‐induced maternal hypercholesterolemia is sufficient to enhance fetal lesion formation. More important, maternal hypercholesterolemia or ensuing pathogenic events in the fetus increase postnatal atherogenesis in response to hypercholesterolemia. Maternal treatment with cholesterol‐lowering agents or antioxidants greatly reduces fetal and postnatal atherogenesis, indicating a pathogenic role of lipid peroxidation and a potential involvement of oxidation‐sensitive signaling pathways. Experiments in a murine model showed that differences in arterial gene expression between offspring of normo‐and hypercholesterolemic mothers persist long after birth, supporting the assumption that fetal lesion formation is associated with genetic programming, which may in turn affect postnatal atherogenesis. A better understanding of pathogenic programming events in utero may lead to the identification of genes determining the susceptibility to atherosclerosis and define novel preventive approaches.—Palinski, W., Napoli, C. The fetal origins of atherosclerosis: maternal hypercholesterolemia, and cholesterol‐lowering or antioxidant treatment during pregnancy influence in utero programming and postnatal susceptibility to atherogenesis. FASEB J. 16, 1348–1360 (2002)
The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes[W]
Authors: Chen Christine Y. , Wong Eric I. , Vidali Luis , Estavillo Athena , Hepler Peter K. , Wu Hen-ming , Cheung Alice Y.
Published: September 2002

Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)–tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1–expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H+ concentrations in the apical region of elongating pollen tubes.
A Cysteine-Rich Extracellular Protein, LAT52, Interacts with the Extracellular Domain of the Pollen Receptor Kinase LePRK2[W]
Authors: Tang Weihua , Ezcurra Inés , Muschietti Jorge , McCormick Sheila
Published: September 2002

Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown. Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events required for pollen germination and pollen tube growth. To search for pollen-expressed ligands for pollen receptor kinases, we used the extracellular domains of three pollen-specific receptor kinases of tomato (LePRK1, LePRK2, and LePRK3) as baits in a yeast two-hybrid screen. We identified numerous secreted or plasma membrane–bound candidate ligands. One of these, the Cys-rich protein LAT52, was known to be essential during pollen hydration and pollen tube growth. We used in vivo coimmunoprecipitation to demonstrate that LAT52 was capable of forming a complex with LePRK2 in pollen and to show that the extracellular domain of LePRK2 was sufficient for the interaction. Soluble LAT52 can exist in differently sized forms, but only the larger form can interact with LePRK2. We propose that LAT52 might be a ligand for LePRK2.
Glycinergic inhibition is essential for co‐ordinating cranial and spinal respiratory motor outputs in the neonatal rat
Authors: Dutschmann M. , Paton J. F. R.
Published: August 2002

Eupnoeic breathing in mammals is dependent on the co‐ordinated activity of cranial and spinal motor outputs to both ventilate the lungs and adjust respiratory airflow, which they do by regulating upper‐airway resistance. We investigated the role of central glycinergic inhibition in the co‐ordination of cranial and spinal respiratory motor outflows. We developed an arterially perfused neonatal rat preparation (postnatal age 0–4 days) to assess the effects of blocking glycine receptors with systemically administered strychnine (0.5–1 μM). We recorded respiratory neurones located within the ventrolateral medulla, inspiratory phrenic nerve activity (PNA) and recurrent laryngeal nerve activity (RLNA), as well as dynamic changes in laryngeal resistance. Central recordings of postinspiratory neurones revealed an earlier onset in firing relative to the onset of inspiratory PNA after exposure to strychnine (260 ± 38.9 vs. 129 ± 26.8 ms). After glycine receptor blockade, postinspiratory neurones discharged during the inspiratory phase. Strychnine also evoked a decrease in PNA frequency (from 38.6 ± 4.7 to 30.7 ± 2.8 bursts min−1), but amplitude was unaffected. In control conditions, RLNA comprised inspiratory and postinspiratory discharges; the amplitude of the latter exceeded that of the former. However, after administration of strychnine, the amplitude of inspiratory‐related discharge increased (+65.2 ± 15.2%) and exceeded postinspiratory activity. Functionally this change in RLNA caused a paradoxical, inspiratory‐related glottal constriction during PNA. We conclude that during the first days of life in the rat, glycine receptors are essential for the formation of the eupnoeic‐like breathing pattern as defined by the co‐ordinated activity of cranial and spinal motor inspiratory and postinspiratory activities.
Exercise and gene expression: physiological regulation of the human genome through physical activity
Authors: Booth Frank W. , Chakravarthy Manu V. , Spangenburg Espen E.
Published: August 2002

The current human genome was moulded and refined through generations of time. We propose that the basic framework for physiologic gene regulation was selected during an era of obligatory physical activity, as the survival of our Late Palaeolithic (50 000–10 000 BC) ancestors depended on hunting and gathering. A sedentary lifestyle in such an environment probably meant elimination of that individual organism. The phenotype of the present dayHomo sapiensgenome is much different from that of our ancient ancestors, primarily as a consequence of expressing evolutionarily programmed Late Palaeolithic genes in an environment that is predominantly sedentary. In this sense, our current genome is maladapted, resulting in abnormal gene expression, which in turn frequently manifests itself as clinically overt disease. We speculate that some of these genes still play a role in survival by causing premature death from chronic diseases produced by physical inactivity. We also contend that the current scientific evidence supports the notion that disruptions in cellular homeostasis are diminished in magnitude in physically active individuals compared with sedentary individuals due to the natural selection of gene expression that supports the physically active lifestyle displayed by our ancestors. We speculate that genes evolved with the expectation of requiring a certain threshold of physical activity for normal physiologic gene expression, and thus habitual exercise in sedentary cultures restores perturbed homeostatic mechanisms towards the normal physiological range of the PalaeolithicHomo sapiens. This hypothesis allows us to ask the question of whether normal physiological values change as a result of becoming sedentary. In summary, in sedentary cultures, daily physical activity normalizes gene expression towards patterns established to maintain the survival in the Late Palaeolithic era.
Model simulations of cardiovascular changes at the onset of moderate exercise in humans
Authors: Elstad Maja , Toska Karin , Walløe Lars
Published: August 2002

We have tested whether the cardiovascular changes at the onset of exercise could be simulated only by an increase in the baroreflex set point and locally induced vasodilatation in the exercising muscles. The mathematical model consists of a heart, a linear elastic arterial reservoir and two parallel resistive vascular beds. The arterial baroreflex loop is modelled by three separate time domain processing objects, each with its own gain, time constant and delay. These are intended to simulate the action of a sympathetic signal to the peripheral vascular bed, a parasympathetic signal to the heart and a sympathetic signal to the heart. We used this model with previously published experimental data to estimate the unknown parameters in the reflex control loop. In all 10 subjects and in the global averaged response, the short‐term cardiovascular responses were adequately simulated by using individual sets of parameters in the model. An increase in the baroreflex set point and locally induced vasodilatation in the exercising muscles can explain almost all of the cardiovascular changes in the recorded variables (mean arterial pressure, RR interval and stroke volume) at the onset of exercise.
Spatial Segregation and Interaction of Calcium Signalling Mechanisms in Rat Hippocampal CA1 Pyramidal Neurons
Authors: Nakamura Takeshi , Lasser‐Ross Nechama , Nakamura Kyoko , Ross William N.
Published: August 2002

Postsynaptic [Ca2+]i increases result from Ca2+ entry through ligand‐gated channels, entry through voltage‐gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca2+ from IP3‐sensitive stores in the form of Ca2+ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 μm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor‐mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca2+]i increases were typically more than eight times larger than [Ca2+]i from this source on the main aspiny apical shaft. Ca2+ entry through voltage‐gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca2+ entry through NMDA receptor channels or voltage‐gated channels (as previously demonstrated) synergistically enhanced Ca2+ release generated by mGluR mobilization of IP3.
Adrenergic signalling between rat taste receptor cells
Authors: Herness Scott , Zhao Fang‐li , Kaya Namik , Lu Shao‐gang , Shen Tiansheng , Sun Xiao‐Dong
Published: August 2002

In taste buds, synaptic transmission is traditionally thought to occur from taste receptor cells to the afferent nerve. This communication reports the novel observation that taste receptor cells respond to adrenergic stimulation. Noradrenaline application inhibited outward potassium currents in a dose‐dependent manner. This inhibition was mimicked by the β agonist isoproterenol and blocked by the β antagonist propranolol. The α agonists clonidine and phenylephrine both inhibited the potassium currents and elevated intracellular calcium levels. Inwardly rectifying potassium currents were unaffected by adrenergic stimulation. Experiments using the RT‐PCR technique demonstrate that lingual epithelium expresses multiple α (α1a, α1b, α1c, α1d, α2a, α2b, α2c) and β (β1, β2) subtypes of adrenergic receptors, and immunocytochemistry localized noradrenaline to a subset of taste receptor cells. Collectively, these data imply strongly that adrenergic transmission within the taste bud may play a paracrine role in taste physiology.
Passive transfer of Lambert‐Eaton syndrome to mice induces dihydropyridine sensitivity of neuromuscular transmission
Authors: Flink Michael T. , Atchison William D.
Published: August 2002

Lambert‐Eaton myasthenic syndrome (LEMS) is a paraneoplastic disorder in which autoantibodies apparently target the voltage‐gated Ca2+ channels that regulate acetylcholine (ACh) release at motor nerve terminals. P/Q‐type Ca2+ channels are primarily involved in ACh release at mammalian neuromuscular junctions. Passive transfer of LEMS to mice by repeated administration of plasma from LEMS patients reduces the amplitude of the perineurial P/Q‐type current, and unmasks a dihydropyridine (DHP)‐sensitive L‐type Ca2+ current at the motor nerve terminal. The present study sought to determine if this DHP‐sensitive component contributes to ACh release. Mice were treated for 30 days with plasma from healthy human controls or patients with LEMS. For some studies, diaphragms from naive mice were incubated with LEMS or control human plasma for 2 or 24 h. End‐plate potentials (EPPs) and miniature end‐plate potentials (MEPPs) were recorded from neuromuscular junctions in the hemidiaphragm. Treatment of mice with LEMS plasma evoked the characteristic electrophysiological signs of LEMS: reduced quantal content and facilitation of EPP amplitudes at high‐frequency stimulation. Quantal content was also reduced in muscles incubated acutely with LEMS plasma. Nimodipine, a DHP‐type blocker of L‐type Ca2+ channels, did not significantly affect the quantal content of muscles treated for 2 or 24 h with either control or LEMS plasma, or following chronic treatment with control plasma. However, following 30 days treatment with LEMS plasma, nimodipine significantly reduced the remaining quantal content to 57.7 ± 3.3% of pre‐nimodipine control levels. Thus, DHP‐sensitive Ca2+ channels become involved in synaptic transmission at the mouse neuromuscular junction after chronic, but not acute treatment with LEMS plasma. However, reductions in quantal release of ACh occur even after very short periods of exposure to LEMS plasma. As such, development of the L‐type Ca2+ channel contribution to ACh release during passive transfer of LEMS appears to occur only after quantal release is significantly impaired for an extended duration, suggesting that an adaptive response of the ACh release apparatus occurs in LEMS.
Brainstem modulation of visual response properties of single cells in the dorsal lateral geniculate nucleus of cat
Authors: Fjeld I.T. , Ruksenas O. , Heggelund P.
Published: August 2002

The dorsal lateral geniculate nucleus (dLGN) transmits visual signals from the retina to the cortex. In the dLGN the antagonism between the centre and the surround of the receptive fields is increased through intrageniculate inhibitory mechanisms. Furthermore, the transmission of signals through the dLGN is modulated in a state‐dependent manner by input from various brainstem nuclei including an area in the parabrachial region (PBR) containing cholinergic cells involved in the regulation of arousal and sleep. Here, we studied the effects of increased PBR input on the spatial receptive field properties of cells in the dLGN. We made simultaneous single‐unit recordings of the input to the cells from the retina (S‐potentials) and the output of the cells to the cortex (action potentials) to determine spatial receptive field modifications generated in the dLGN. State‐dependent modulation of the spatial receptive field properties was studied by electrical stimulation of the PBR. The results showed that PBR stimulation had only a minor effect on the modifications of the spatial receptive field properties generated in the dLGN. The PBR‐evoked effects could be described mainly as increased response gain. This suggested that the spatial modifications of the receptive field occurred at an earlier stage of processing in the dLGN than the PBR‐controlled gain regulation, such that the PBR input modulates the gain of the spatially modified signals. We propose that the spatial receptive field modifications occur at the input to relay cells through the synaptic triades between retinal afferents, inhibitory interneurone dendrites, and relay cell dendrites and that the gain regulation is related to postsynaptic cholinergic effects on the relay cells.
Combined inhibition of nitric oxide and prostaglandins reduces human skeletal muscle blood flow during exercise
Authors: Boushel Robert , Langberg Henning , Gemmer Carsten , Olesen Jens , Crameri Regina , Scheede Celena , Sander Michael , Kjær Michael
Published: August 2002

The vascular endothelium is an important mediator of tissue vasodilatation, yet the role of the specific substances, nitric oxide (NO) and prostaglandins (PG), in mediating the large increases in muscle perfusion during exercise in humans is unclear. Quadriceps microvascular blood flow was quantified by near infrared spectroscopy and indocyanine green in six healthy humans during dynamic knee extension exercise with and without combined pharmacological inhibition of NO synthase (NOS) and PG by l‐NAME and indomethacin, respectively. Microdialysis was applied to determine interstitial release of PG. Compared to control, combined blockade resulted in a 5‐ to 10‐fold lower muscle interstitial PG level. During control incremental knee extension exercise, mean blood flow in the quadriceps muscles rose from 10 ± 0.8 ml (100 ml tissue)−1 min−1 at rest to 124 ± 19, 245 ± 24, 329 ± 24 and 312 ± 25 ml (100 ml tissue)−1 min−1 at 15, 30, 45 and 60 W, respectively. During inhibition of NOS and PG, blood flow was reduced to 8 ± 0.5 ml (100 ml tissue)−1 min−1 at rest, and 100 ± 13, 163 ± 21, 217 ± 23 and 256 ± 28 ml (100 ml tissue)−1 min−1 at 15, 30, 45 and 60 W, respectively (P < 0.05 vs. control). In conclusion, combined inhibition of NOS and PG reduced muscle blood flow during dynamic exercise in humans. These findings demonstrate an important synergistic role of NO and PG for skeletal muscle vasodilatation and hyperaemia during muscular contraction.
An External Site Controls Closing of the Epithelial Na+ Channel ENaC
Authors: Kellenberger Stephan , Gautschi Ivan , Schild Laurent
Published: August 2002

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid‐sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the ‘degenerin site’ activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (βS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a ∼3‐fold increase in the open probability. This effect is due to an 8‐fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N‐terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of βS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state‐ and size‐dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.
A‐Type potassium currents active at subthreshold potentials in mouse cerebellar purkinje cells
Authors: Sacco Tiziana , Tempia Filippo
Published: August 2002

Voltage‐dependent and calcium‐independent K+currents were whole‐cell recorded from cerebellar Purkinje cells in slices. Tetraethylammonium (TEA, 4 mm) application isolated an A‐type K+current (Ik(a)) with a peak amplitude, at +20 mV, of about one third of the total voltage‐dependent and calcium‐independent K+current. TheIk(a)activated at about −60 mV, had aV0.5of activation of −24.9 mV and aV0.5of inactivation of −69.2 mV. The deactivation time constant at −70 mV was 3.4 ± 0.4 ms, while the activation time constant at +20 mV was 0.9 ± 0.2 ms. The inactivation kinetics was weakly voltage dependent, with two time constants; those at +20 mV were 19.3 ± 3.1 and 97.6 ± 9.8 ms. The recovery from inactivation had two time constants of 60.8 ms (78.4%) and 962.3 ms (21.6%). TheIk(a)was blocked by 4‐aminopyridine with an IC50of 67.6 μM. Agitoxin‐2 (2 nm) blocked 17.4 ± 2.1% of theIk(a). Flecainide completely blocked theIk(a)with a biphasic effect with IC50values of 4.4 and 183.2 μM. In current‐clamp recordings the duration of evoked action potentials was affected neither by agitoxin‐2 (2 nm) nor by flecainide (3 μM), but action potentials that were already broadened by TEA were further prolonged by 4‐aminopyridine (100 μM). The amplitude of the hyperpolarisation at the end of depolarising steps was reduced by all these blockers.
Isotonic force modulates force redevelopment rate of intact frog muscle fibres: evidence for cross‐bridge induced thin filament activation
Authors: Vandenboom Rene , Hannon James D. , Sieck Gary C.
Published: August 2002

We tested the hypothesis that force‐velocity history modulates thin filament activation, as assessed by the rate of force redevelopment after shortening (+dF/dtR). The influence of isotonic force on +dF/dtR was assessed by imposing uniform amplitude (2.55 to 2.15 μm sarcomere−1) but different speed releases to intact frog muscle fibres during fused tetani. Each release consisted of a contiguous ramp‐ and step‐change in length. Ramp speed was changed from release to release to vary fibre shortening speed from 1.00 (2.76 ± 0.11 μm half‐sarcomere−1 s−1) to 0.30 of maximum unloaded shortening velocity (Vu), thereby modulating isotonic force from 0 to 0.34 Fo, respectively. The step zeroed force and allowed the fibre to shorten unloaded for a brief period of time prior to force redevelopment. Although peak force redevelopment after different releases was similar, +dF/dtR increased by 81 ± 6% (P < 0.05) as fibre shortening speed was reduced from 1.00 Vu. The +dF/dtR after different releases was strongly correlated with the preceding isotonic force (r= 0.99, P < 0.001). Results from additional experiments showed that the slope of slack test plots produced by systematically increasing the step size that followed each ramp were similar. Thus, isotonic force did not influence Vu (mean: 2.84 ± 0.10 μm half‐sarcomere−1 s−1, P < 0.05). We conclude that isotonic force modulates +dF/dtR independent of change in Vu, an outcome consistent with a cooperative influence of attached cross‐bridges on thin filament activation that increases cross‐bridge attachment rate without alteration to cross‐bridge detachment rate.
The presence of ca2+ channel β subunit is required for mitogen‐activated protein kinase (mapk)‐dependent modulation of α1b ca2+ channels in cos‐7 cells
Authors: Fitzgerald Elizabeth M.
Published: August 2002

In rat sensory neurones, voltage‐dependent calcium channels (VDCCs), including the N‐type, are tonically up‐regulated via Ras/mitogen‐activated protein kinase (MAPK) signalling. To determine whether VDCC β subunit is involved in this process, the role of the four neuronal βs (β1b, β2a, β3, β4) in MAPK‐dependent modulation of α1B (Cav2.2, N‐type) Ca2+ channels has been examined in COS‐7 cells. MAPK is exclusively activated by MAPK kinase (MEK), and here, acute application of a MEK‐specific inhibitor UO126, significantly inhibited peak α1B Ca2+ channel current (Imax) within a period of 5–10 min, regardless of which β subunit was co‐expressed (25‐50%, P < 0.01). With β2a however, the percentage inhibition of Imax was less than that observed with any other β (ANOVA: F3,34= 6.48, P < 0.01). UO126 also caused a hyperpolarising shift (6 ± 1 mV, P < 0.001) in the voltage dependence of β2a current activation, such that inhibition occurred only at depolarised potentials (> +5 mV) whereas at more negative potentials the current amplitude was enhanced. A marked change in β2a current kinetics, perceived either as decreased activation or increased inactivation, was also associated with UO126 application. A similar effect of UO126 on β4 current kinetics was also observed. The β2a‐specific effects of UO126 on current inhibition and voltage dependence of activation were abolished when α1B was co‐expressed with de‐palmitoylated β2a(C3,4S), in which amino terminal cysteines 3 and 4 had been mutated to serines. In the absence of β subunit, UO126 had no effect on α1B Ca2+ channel current. Together, these data suggest an absolute requirement for β in MAPK‐dependent modulation of these channels. Since β subunits vary both in their temporal expression and localisation within neurones, β subunit‐dependent modulation of N‐type Ca2+ channels via MAPK could provide an important new mechanism by which to fine‐tune neurotransmitter release.
Mechanisms of acid‐induced activation of airway afferent nerve fibres in guinea‐pig
Authors: Kollarik Marian , Undem Bradley J.
Published: August 2002

The mechanisms underlying the response of airway afferent nerves to low pH were investigated in an isolated guinea‐pig airway nerve preparation. Extracellular recordings were made from single jugular or nodose vagal ganglion neurons that projected their sensory fibers into the airways. The airway tissue containing the mechanically sensitive receptive fields was exposed into acidic solutions. Rapid and transient (∼3 s) administration of 1 mm citric acid to the receptive field consistently induced action potential discharge in nociceptive C‐fibers (41/44) and nodose Aδ fibres (29/30) that are rapidly adapting low threshold mechanosensors (RAR‐like fibres). In contrast, citric acid activated only 8/17 high threshold mechanosensitive jugular Aδ fibres. The RAR‐like fibres were slightly more sensitive than C‐fibres to acidic solutions (pH threshold > 6.7). The RAR‐like fibres response to the ∼3 s acid treatment was not affected by a vanilloid receptor 1 (VR1) antagonist, capsazepine (10 μM), and was rapidly inactivating (action potential discharge terminated before the acid administration was completed). Gradual reduction of pH did not activate the RAR‐like fibres even when the pH was reduced to ∼5.0. The C‐fibres responded to the gradual reduction of pH with persistent action potential discharge that was nearly abolished by capsazepine (10 μM) and inhibited by over 70% with another VR1 antagonist iodo‐resiniferatoxin (1 μM). In contrast the C‐fibre response to the transient ∼3 s exposure to pH ∼5.0 was not affected by the VR1 antagonists. We conclude that activation of guinea‐pig airway afferents by low pH is mediated by both slowly and rapidly inactivating mechanisms. We hypothesize that the slowly inactivating mechanism, present in C‐fibres but not in RAR‐like fibres, is mediated by VR1. The rapidly inactivating mechanism acts independently of VR1, has characteristics similar to acid sensing ion channels (ASICs) and is found in the airway terminals of both C‐fibres and RAR‐like fibres.