ß-Scorpion Toxin Modifies Gating Transitions in All Four Voltage Sensors of the Sodium Channel

doi:10.1085/jgp.200609719The Journal of General Physiology

Fabiana V. Campos1,2, Baron Chanda3, Paulo S.L. Beirão2, and Francisco Bezanilla1

Several naturally occurring polypeptide neurotoxins target specific sites on the voltage-gated sodium channels. Of these, the gating modifier toxins alter the behavior of the sodium channels by stabilizing transient intermediate states in the channel gating pathway. Here they have used an integrated approach that combines electrophysiological and spectroscopic measurements to determine the structural rearrangements modified by the ß-scorpion toxin Ts1. Our data indicate that toxin binding to the channel is restricted to a single binding site on domain II voltage sensor. Analysis of Cole-Moore shifts suggests that the number of closed states in the activation sequence prior to channel opening is reduced in the presence of toxin. Measurements of charge–voltage relationships show that a fraction of the gating charge is immobilized in Ts1-modified channels. Interestingly, the charge–voltage relationship also shows an additional component at hyperpolarized potentials. Site-specific fluorescence measurements indicate that in presence of the toxin the voltage sensor of domain II remains trapped in the activated state. Furthermore, the binding of the toxin potentiates the activation of the other three voltage sensors of the sodium channel to more hyperpolarized potentials. These findings reveal how the binding of ß-scorpion toxin modifies channel function and provides insight into early gating transitions of sodium channels.

AHSP: a novel hemoglobin helper

J Clin Invest. 2007 July 2; 117(7): 1746–1749.
Published online 2007 July 2. doi: 10.1172/JCI32362.

Arthur Bank
Department of Medicine and Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York, USA.

Recently, the small protein α hemoglobin–stabilizing protein (AHSP) was identified and found to specifically bind α-globin, stabilize its structure, and limit the toxic effects of excess α-globin, which are manifest in the inherited blood disorder β thalassemia. In this issue of the JCI, Yu, Weiss, and colleagues show that AHSP is also critical to the formation and stabilization of normal amounts of hemoglobin, even when α-globin is deficient, indicating unique and previously unidentified roles for this molecule

Now, in this issue of the JCI (11), Yu, Weiss, and colleagues show that AHSP is a much more adroit Hb helper, facilitating even more important new “twists” in Hb assembly. The authors show that AHSP is important not only for dealing with newly synthesized excess α-globin, but also in the assembly of normal Hb tetramers. In these new studies, Ahsp–/– mice with mild α thalassemia were examined. This condition is associated with a deficit of α-globin and an excess of β-globin, so no specific role for AHSP was expected to be required in these mice. However, Ahsp–/– mice with α thalassemia were found to be more anemic than either Ahsp–/– mice or α thalassemic mice. If the role of AHSP is only to stabilize apo-α-globin and αHb, why should its absence have any effect?
The authors provide some of the possible answers (11). They show that the anemia in the Ahsp–/– mice with α thalassemia is accompanied by an excess of precipitated β-globin chains in the membrane of erythroid cells (Figure 1C), which are present in much greater quantities than in either Ahsp–/– mice or α thalassemic mice. Thus, AHSP is required for the assembly of normally synthesized excess β chains into functional HbA in these mice. Even the presence of small amounts of newly synthesized apo-α-globin and αHb not stabilized by AHSP results in the inability of excess β-globin chains to be efficiently transferred into HbA in these α thalassemic mice. Thus, without AHSP, excess β-globin and βHb are also unstable and deposited in the red cell membrane, leading to increased oxidative damage and more severe anemia (Figure 1C). The authors show that levels of ROS are greatly increased in the double mutants as compared with single mutants, reflecting this pathology (11). They also demonstrate that the deleterious effects of AHSP deficiency increase in marrow nucleated erythroid cell populations as they accumulate more globin and Hb, confirming and extending previous results (12).
Potential roles for AHSP as an intermediate in the optimal formation of normal Hbαβ dimers and/or tetramers remain to be determined. It is clear from this study (11), however, that AHSP is necessary for more than just chaperoning α-globin around: it is also necessary for normal HbA assembly, especially when there is an imbalance in either α- or β-globin.
The results of several elegant in vitro experiments in this study (11) also provide new details regarding the way AHSP handles α-globin in cells. Newly synthesized α-globin chains, even nascent α-globin chains on ribosomes, are rapidly complexed to AHSP in cell-free systems. More HbA is formed in the presence of AHSP than without it. AHSP increases the resistance of apo-α-globin to proteolysis by trypsin by promoting the proper folding of α-globin in the αHb-AHSP complex. Even after denaturation of apo-α-globin chains, their renaturation is shown to be strongly promoted by the presence of AHSP (11).

In summary, AHSP has been identified as a unique Hb helper, a molecular chaperone required for normal Hb assembly. Yu, Weiss, and colleagues make the interesting suggestion that AHSP provides a selective advantage for the survival of red cells, especially when there are significant amounts of either excess α- or β-globin present (11). Interestingly, the red cells of patients with α and β thalassemia are more resistant to the severe form of malaria than normal cells (13). The evolution of AHSP may have permitted the preferential survival of these cells. Without AHSP, the thalassemic red cells might not have survived, while with it, they are able to. Thus, AHSP may have evolved to give erythroid progenitors an “edge,” especially when mutations occur that lead to significantly unbalanced α- or β-globin levels. Then, throughout evolution, the AHSP-expressing cells with globin mutations may have been further selected to survive because these cells prevented fatal malarial infection.
Also, because of its effects on preventing α-globin denaturation and promoting renaturation, AHSP may provide an additional selective advantage to red cells under conditions of oxidative stress induced by drugs that cause a greater susceptibility to hemolysis. AHSP may also be useful to red cells in iron deficiency in which heme availability is limited and apo-α-globin levels are increased. These functions may represent additional evolution-based roles for AHSP in the stabilization of red cells in the presence of environmental factors that alter Hb’s critical equilibrium. No human disease resembling AHSP deficiency has yet been described, although associations between the severity of β thalassemia in patients with variations in AHSP are being explored (14, 15). In a case of a naturally occurring human α-globin chain mutation, in which the binding site for αHb-AHSP complex formation is altered, there is decreased stability of the resulting human Hb (16).
Are there other Hb helpers, be they specific α-globin chaperones or other erythroid-specific or ubiquitous molecules, with which AHSP interacts? It is known that the transcription factors GATA-1, OCT-1, and EKLF are required for AHSP expression (17, 18). How does AHSP interact with these and other transcription factors and intermediates affecting heme biosynthesis and posttranscriptional modifiers in red cells in the process of Hb synthesis and assembly? These and other questions regarding our understanding of Hb regulation remain. We are also still in the dark about what controls the differentiation of nucleated red cells and their enucleation, what regulates the filling of red cells with the desired amount of Hb, and how that amount is maintained until red cell death. Does AHSP function in these events? Weiss et al. (11) have given us a start by identifying an important Hb helper, but there is plenty of room for researchers to discover other Hb helpers and to shed more light on this subject.

Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs)

J Clin Invest. 2007 July 2; 117(7): 1926–1932.
Published online 2007 June 7. doi: 10.1172/JCI31370.
Carole Peyssonnaux,1,2 Annelies S. Zinkernagel,2 Reto A. Schuepbach,3 Erinn Rankin,4 Sophie Vaulont,5,6 Volker H. Haase,4 Victor Nizet,2 and Randall S. Johnson1
Iron is essential for many biological processes, including oxygen delivery, and its supply is tightly regulated. Hepcidin, a small peptide synthesized in the liver, is a key regulator of iron absorption and homeostasis in mammals. Hepcidin production is increased by iron overload and decreased by anemia and hypoxia; but the molecular mechanisms that govern the hepcidin response to these stimuli are not known. Here we establish that the von Hippel–Lindau/hypoxia-inducible transcription factor (VHL/HIF) pathway is an essential link between iron homeostasis and hepcidin regulation in vivo. Through coordinate downregulation of hepcidin and upregulation of erythropoietin and ferroportin, the VHL-HIF pathway mobilizes iron to support erythrocyte production.Hepcidin is suppressed by both anemia and hypoxia (7). Cellular oxygen sensing and hypoxia-induced transcription are largely mediated by the HIFs. The stabilization of HIF-1 by iron chelators has been well established in vitro. However, our demonstration of the in vivo induction of HIF-1 by iron deficiency, and the associated downregulation of hepcidin when HIF levels are elevated, suggests that HIF may be one of the missing links between iron homeostasis and hepcidin regulation. The ability of HIF-1α to bind to and negatively transactivate the hepcidin promoter suggests a direct repressor effect. HIF-1α has already been reported to repress the transcriptional activity of genes such as alpha-fetoprotein (AFP) (28) and carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) (29).
Our data show that elimination of HIF-1 alone in adult mice is insufficient to fully compensate for the hepcidin reduction induced by iron depletion. The striking downregulation of hepcidin observed in Albumin-Cre/VHLflox/flox mice, in which both HIF-1 and HIF-2 are stabilized, may suggest a role for HIF-2. HIF-1 may influence embryonic, developmental hepcidin regulation; as Yoon et al. demonstrated, a decrease in TfR in Hif-1a–/– embryos contributes to defects in iron metabolism and consequently an alteration of hepcidin levels (30).
We propose that the VHL/HIF axis serves a central role in coupling iron sensing to iron regulation. In this model, anemia causes decreased tissue oxygenation, which in turn leads to decreased PHD activity and thus decreased VHL-mediated degradation of HIF-α factors. Increased HIF-α activity causes suppression of hepcidin, increased ferroportin levels, and increased serum iron availability; these in turn allow increased erythropoiesis to be coupled to increased EPO expression.
In support of a role for the HIF axis in regulating this process, we have shown that hepatic deletion of the VHL gene causes decreased hepcidin levels and increased ferroportin expression. Interestingly, in the face of dramatically increased EPO levels and polycythemia, the VHL-deficient animals exhibited microcytosis and low MCH reminiscent of iron-deficiency. This finding suggests a form of “iron-EPO kinetic imbalance,” where the robust proliferation of erythroid precursors creates a demand that outstrips the capacity of the iron delivery system (31). Our model also suggests that inhibition of VHL or PHD activity could represent a novel approach for treatment of anemia of chronic inflammation, since stabilization of HIF prevents hepcidin activation even under the strong stimulus of the proinflammatory cytokine IL-6.
It has been proposed that iron balance is conceptually coupled to intestinal iron absorption by 2 mechanisms, the “stores regulator” and the “erythroid regulator,” which can be fully characterized only in physiological terms (32). Our results indicate the VHL/HIF axis concomitantly serves both the stores and erythroid regulator pathways, through its responsiveness to oxygen and iron levels and its function as a regulator of hepcidin, ferroportin, and EPO production.

The renin-angiotensin system: it’s all in your head

J Clin Invest. 2007 April 2; 117(4): 873–876.
doi: 10.1172/JCI31856

Kelly K. Parsons and Thomas M. Coffman
Division of Nephrology, Department of Medicine, Duke University School of Medicine, and Durham Veterans Affairs Medical Center, Durham, North Carolina, USA.

Components of the renin-angiotensin system (RAS) are expressed in a number of areas in the brain involved in cardiovascular control. However, it has been difficult to link RAS actions in circumscribed brain regions to specific physiological functions. In a study appearing in this issue of the JCI, Sakai and associates use a combination of sophisticated transgenic techniques and stereotaxic microinjection of recombinant viral vectors to demonstrate a pivotal role in the regulation of thirst and salt appetite of angiotensin II generated in the subfornical organ in the brain.The capacity of the CNS to respond directly to angiotensin II was demonstrated in experiments more than 2 decades ago, wherein angiotensin II was injected into cerebral ventricles or specific brain nuclei and it subsequently elicited potent cardiovascular and dipsogenic (thirst-provoking) responses (6, 7). Expression of virtually all of the components of the RAS has since been verified in various regions and cell lineages in the brain. Based on these findings, it was suggested that angiotensin II generated locally in the brain might function as a putative neurotransmitter in neurons involved in cardiovascular regulation (8). Within the brain, the subfornical organ (SFO) is a major site of RAS activity that has also been implicated as an important cardiovascular control center. Injection of angiotensin II into the SFO activates its neurons and elicits potent systemic vasoconstriction . While angiotensinogen and angiotensin receptors are highly expressed within the SFO (10–14), it lies outside of the blood-brain barrier and therefore is also potentially subject to modulation by components of the RAS in the circulation.
Many of the general features of the brain RAS have been apparent for some time. Until recently, however, it has been difficult to develop a precise understanding of its contributions to physiological homeostasis in the intact organism. This was due to a number of experimental barriers, including gaps in expression patterns of RAS components between regions and difficulties in precisely manipulating the brain RAS in vivo. The development of techniques for transgenesis and gene targeting advanced the field, but the lack of promoters to drive expression in specific brain regions remains a significant limitation. In the current study by Sakai and associates (4), along with another recent publication by the same group (5), transgenic mouse lines expressing RAS genes have been utilized in combination with microinjection of recombinant viral vectors to establish a critical contribution of angiotensin II generation within the SFO to the control of thirst and systemic vasopressor responses

Regulation of erythrocyte lifespan: do reactive oxygen species set the clock?

J Clin Invest. 2007 August 1; 117(8): 2075–2077.
doi: 10.1172/JCI32559.
Shilpa M. Hattangadi1,2 and Harvey F. Lodish1
1Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. 2Children’s Hospital of Boston, Boston, Massachusetts, USA.
Red blood cell homeostasis is an excellent example of redox balance: erythroid progenitors accumulate hemoglobin during development, and erythrocytes continuously transport large amounts of oxygen over the course of their approximately 120-day lifespan. This results in a high level of oxidative stress (1). Fully mature red blood cells, lacking a nucleus, cannot produce new proteins in response to stress — they have to rely on proteins synthesized earlier in development to protect themselves from damage by ROS and thus ensure their own survival. Red blood cells have thus evolved to have an extensive array of antioxidants to counter this level of stress, including membrane oxidoreductases, cellular antioxidants such as catalase and superoxide dismutase (SOD), and enzymes that continuously produce reducing agents through the glutathione (GSH) system (2).
Defects in enzymes critical to the oxidative stress response have been implicated in human diseases ranging from mild chronic hemolysis to severe acute hemolysis (2). Because of a concomitant reduction in the normal red blood cell lifespan, these disease states are characterized by a compensatory increase in erythropoiesis, evident in patients as reticulocytosis. One relatively common genetic disorder is deficiency of glucose-6-phosphate dehydrogenase (G6PD), the enzyme that converts NADP to NADPH. NADPH is required for the maintenance of reduced GSH, and GSH in turn reduces peroxides, superoxides, and other ROS (2). G6PD is therefore required to protect the red blood cell from oxidative damage, and absence of this protection can result in severe hemolysis.
The deleterious effects of oxidative stress, such as damage to cellular proteins, DNA, and lipids, are well characterized. The dependence of the lifespan of the erythrocyte on an adequate antioxidant response has been previously demonstrated (3). However, the factors that regulate the oxidative stress response and the lifespan of erythrocytes are less clear.

Marinkovic et al. (10) suggest that the mitotic arrest observed in Foxo3-null Ter119+CD71+ erythrocyte precursors may be caused by p53-dependent G1-phase arrest induced as a response to stress through its downstream target, p21CIP1/WAF1/Sdi1, as both genes were upregulated in Foxo3-deficient erythroid precursors. There was also induction of the antioxidant p53 downstream targets GADD45 and sestrin 2 (SESN2). The authors speculate that the oxidative stress left unchecked by lack of Foxo3 likely turns on the p53 pathway in erythroid progenitors, resulting in induction of downstream targets that may mitigate oxidative stress and activate resistance to ROS-mediated damage.
Another interesting observation by Marinkovic et al. (10) was that the mitotic arrest and induction of p21CIP1/WAF1/Sdi1 in intermediate Foxo3-null erythroid progenitors was improved with NAC treatment. This raises the interesting possibility that ROS levels regulate Foxo3 or at least its function of initiating the oxidative stress response, creating a type of erythroid differentiation checkpoint. Hypothetically, when sufficient hemoglobin has been produced for the nascent terminal erythrocyte to carry out its oxygen-carrying tasks, the resulting oxidative stress from oxygen and heme moieties would somehow trigger Foxo3 translocation to the nucleus and induction of its targets in terminal differentiation. This as yet untested hypothesis emphasizes the importance of determining both the direct targets of Foxo3 and its own regulation in helping us understand how a red blood cell lacking a nucleus knows exactly when to die.

Effect of ACE inhibition on skeletal muscle oxidative function and exercise capacity in streptozotocin-induced diabetic rats

Experimental Physiology (2007)DOI: 10.1113/expphysiol.2007.038851
Olivier Rouyer 1, Joffrey Zoll 1, Frederic Daussin 1, Christiane Damge 2, Pauline Helms 1, Samy Talha 1, Laurence Rasseneur 3, Francois Piquard 2, Bernard Geny 1*1 Physiologie et Explorations Fonctionnelles2 Institut de Physiologie3 STAPS
Since exercise capacity relates to the mitochondrial respiration rate in skeletal muscle and both parameters are potentially modulated by the onset of diabetes and by inhibition of the angiotensin-converting enzyme (ACE), we investigated whether skeletal muscle oxidative functions and exercise capacities are impaired in chronic streptozotocin-induced diabetic (STZ) rats and whether ACE inhibition could reverse such abnormalities. The ACE inhibitor perindopril (2mg/kg/day) was given for a period of 5 weeks to seven month old STZ rats (DIA-PE, n=8) whose hemodynamic, skeletal muscle mitochondrial function and exercise capacity were compared to that of untreated diabetic (DIA, n=8) and control (CONT, n=8) rats. Increased arterial blood pressureand reduced exercise capacitywere observed in DIA compared to CONT.The oxidative capacity of the gastrocnemius muscle was significantly reduced in DIA compared to CONT ratsMoreover, the coupling between oxidation and phosphorylation was significantly impaired in DIA . ACE inhibition (ACEi) normalized blood pressure without improving mitochondrial function but actually reduced exercise capacity to even lower levels.Exercise capacity correlated positively with blood pressure in DIA-PE
In experimental type 1 diabetic rats, both skeletal muscle mitochondrial respiration and exercise capacity are impaired. ACEi failed to restore the muscular function and worsened exercise capacity. Further studies will be useful to determine whether an inadequate muscular blood flow secondary to the mean systemic blood pressure reduction can explain these results.
(N.B/Reference of the picture: Essentials of Human Physiology by Thomas M. Nosek, Ph.D)

Pharmacogenomics of Neuroimmuno Interactions in Psychiatric Disorders

Experimental Physiology (2007)DOI: 10.1113/expphysiol.2007.038471
Julio Licinio 1*, Claudio Mastronardi 1, Ma-Li Wong 11 University of Miami* To whom correspondence should be addressed. E-mail: licinio@miami.edu

There is bidirectional communication between the brain and the immune system. Overproduction of interleukin-1-beta (IL-1 ) leads to systemic inflammatory response syndrome (SIRS). The crucial role of IL-1 in inflammation has been highlighted by studies performed in caspase-1 knockout mice (casp1-/-), transgenic mice that lack mature IL-1 and survive lethal doses of lypopolysaccharide (LPS). We have previously shown that IL-1 , its receptor IL-1 receptor I (IL-1RI) and casp1 are expressed within the brain. Moreover, we documented that peripherally-injected LPS triggers a specific spatial-temporal pattern of expression of IL-1 mRNA within the brain suggesting that IL-1 could be a major regulator of the central inflammatory cascade. Therefore, we studied brain transcriptional patterns that occur during LPS-induced SIRS in wild-type and casp1-/- mice. We showed patterns of gene expression in wildtype and casp1-/- mice that included differential expression several genes such as cytokines, chemokines, NOS2 and COX-2. A key component of the neurommune-endocrine axis that is increased by IL-1 is corticotropin-releasing hormone (CRH). We found increased response to antidepressants in patients homozygous for the GAG haplotype of CRH receptor-1. Our results support the hypotheses that the CRH receptor-1 gene and possibly other genes in stress-inflammatory pathways are involved in response to antidepressant treatment. As dysregulation of the neruoimmuno-endocrine axis appears part of the fundamental biological mechanisms that underlie psychiatric disorders, our findings might contribute increase the understanding of the molecular pathways that are altered in these diseases.
(N.B/Reference of the picture: Essentials of Human Physiology by Thomas M. Nosek, Ph.D.)

The vasodilator 17,18-Epoxyeicosatetraenoic acid targets the pore-forming BK channel subunit

Experimental Physiology (2007)DOI: 10.1113/expphysiol.2007.038166Hantz C Hercule 1, Birgit Salanova 1, Kirill Essin 2, Honeck Horst 3, John R Falck 4, Matthias Sausbier 5, Peter Rut 5, Wolf H Schunck 6, Friedrich C Luft 1, Maik Gollasch 2*To whom correspondence should be addressed. E-mail: maik.gollasch@charite.de
17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large-conductance K+ (BK) channels. BK channels are composed of the pore-forming BK and auxiliary BK ß1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine-sensitive, calcium-release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole-cell and perforated-patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague-Dawley rats, BK 1-deficient (-/-) mice, BK (-/-), RyR3 (-/-) mice, and wild-type mice. 17,18-EETeTr (100 nM) increased tetraethylammonium (1 mM)-sensitive outward K+ currents in VSMC from wild-type rats and wild-type mice. The effects were not inhibited by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 µM). BK channel currents were increased 3.5-fold in VSMC from BK 1 (-/-) mice whereas a 2.9-fold stimulation was observed in VSMC from RyR3 (-/-) mice (VM=60 mV). The effects were similar compared to those observed in cells from wild-type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca2+, 100 nM), nor by external calcium influx. 17,18-EETeTr did not induce outward currents in VSMC BK (-/-) cells. We next tested the vasodilator effects of 17,18-EETeTr on isolated arteries of BK -deficient mice. Vasodilation was largely inhibited in cerebral and mesenteric arteries isolated from BK (-/-) mice, compared to those observed in wild-type and BK 1 (-/-) arteries. We conclude that 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. Because 17,18-EETeTr is active in small arteries lacking BK 1, the data further suggest that BK represents the molecular target for 17,18-EETeTr's principal action. Finally, the action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Chronic effects of type 2 diabetes mellitus on cardiac muscle contraction in the Goto-Kakizaki rat

Experimental Physiology (2007)DOI: 10.1113/expphysiol.2007.038703
Frank Christopher Howarth 1*, Mohamed Shafiullah 1, Mohamed Anwar Qureshi 11 United Arab Emirates University* To whom correspondence should be addressed. E-mail: chris.howarth@uaeu.ac.ae

Type 2 diabetes mellitus (DM) accounts for more than 90 % of all cases of DM and cardiovascular complications are the major cause of mortality and death in diabetic patients. The chronic effects of type 2 DM on heart function have been investigated in the Goto-Kakizaki (GK) rat. Experiments were performed in GK rats and age-matched Wistar controls at 18 months of age. The progressive effects of diabetes on glucose metabolism were monitored periodically by application of the glucose tolerance test. Ventricular action potentials were measured in isolated perfused heart, shortening and intracellular Ca2+ were measured in electrically stimulated ventricular myocytes. GK rats displayed mild fasting hyperglycaemia and progressively worsening glucose tolerance. At 18 months of age and 180 min after intraperitoneal injection of glucose challenge (2 g / Kg body weight) blood glucose was 436 ± 47 mg/dl in GK rats compared to 153 ± 18 mg/dl in controls. Heart weight : body weight ratio was significantly increased in GK rats (4.10 ± 0.09, n=5) compared to controls (3.36 ± 0.22, n=4). Spontaneous heart rate was slightly reduced in GK rats compared to controls. Although the amplitude of shortening was not altered the amplitude of the Ca2+ transient was significantly increased in myocytes from GK (0.78 ± 0.11 RU) rats compared to controls (0.50 ± 0.06 RU). Despite progressively worsening glucose metabolism, at 18 months of age, the contractile function of the heart appears to be well preserved.
(N.B/Reference of the picture: Essentials of Human Physiology by Thomas M. Nosek, Ph.D)

Signalling within the neurovascular unit in the mammalian retina

Experimental Physiology 92.4 pp 635-640DOI: 10.1113/expphysiol.2006.036376Monica R. Metea1 and Eric A. Newman11 Department of Neuroscience, University of Minnesota, 6-145 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA

Neuronal activity in the central nervous system evokes localized changes in blood flow, a response termed neurovascular coupling or functional hyperaemia. Modern functional imaging methods, such as functional magnetic resonance imaging (fMRI), measure signals related to functional hyperaemia in order to determine localization of brain function and to diagnose disease. The cellular mechanisms that underlie functional hyperaemia, however, are not well understood. Glial cells have been hypothesized to be intermediaries between neurons and blood vessels in the control of neurovascular coupling, owing to their ability to release vasoactive factors in response to neuronal activity. Using an in vitro preparation of the isolated, intact rodent retina, they have investigated two likely mechanisms of glial control of the vasculature: glial K+ siphoning and glial induction of vasoactive arachidonic acid metabolites. Potassium siphoning is a process by which a K+ current flowing through glial cells transfers K+ released from active neurons to blood vessels. Since slight increases in extracellular K+ can cause vasodilatation, this mechanism was hypothesized to contribute to neurovascular coupling. This suggest that glial K+ siphoning does not contribute significantly to neurovascular coupling in the retina. Instead, they suggest that glial cells mediate neurovascular coupling by inducing the production of two types of arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE), which dilate and constrict vessels, respectively. They show that both light flashes and direct glial stimulation produce vasodilatation or vasoconstriction mediated by EETs and 20-HETE, respectively. Further, they show that the type of vasomotor response observed (dilatation or constriction) depends on retinal levels of nitric oxide. They also demonstrate that glial cells are necessary intermediaries for signalling from neurons to blood vessels, since functional hyperaemia does not occur when neuron-to-glia communication is interrupted. These results indicate that glial cells play an important role in mediating functional hyperaemia and suggest that the regulation of blood flow may involve both vasodilating and vasocostrcting components.

(N.B/Reference of the picture: Essentials of Human Physiology by Thomas M. Nosek, Ph.D)

Behaviours of pulmonary sensory receptors during development of acute lung injury in the rabbit

DOI: 10.1113/expphysiol.2006.036673Shuxin Lin1, Jerome Walker1,2, Ling Xu3, David Gozal4 and Jerry Yu1Departments of 1 Medicine4 Pediatrics, University of Louisville, Louisville, KY 40292, USA 2 Department of Respiratory Therapy, Bellarmine University, Louisville, KY 40205, USA 3Department of Mathematics and Statistics, James Madison University, Harrisonburg, VA 22807, USA
They tested the hypothesis that oleic acid-induced acute lung injury activates pulmonary nociceptors, that is, C fibre receptors (CFRs) and high-threshold A fibre receptors (HTARs). Single-unit activity was recorded in the cervical vagus nerve and assessed before and after injecting oleic acid (75 µl kg–1 I.V.) into anaesthetized, open-chest, mechanically ventilated rabbits. Unit activities increased within seconds and peaked within a few minutes (from 0.3 ± 0.1 to 1.4 ± 0.9 impulses s–1 for CFRs and from 0.5 ± 0.1 to 1.7 ± 0.3 impulses s–1 for HTARs, both n = 8 and P < 0.05). These activities were sustained while pulmonary oedema developed and dynamic lung compliance decreased over the 90 min observation period. Activities in slowly adapting receptors and rapidly adapting receptors were also increased; however, their responsiveness to airway pressure stimulation decreased progressively. We conclude that pulmonary nociceptors are stimulated during acute lung injury. The dual nociceptor system, consisting of both non-myelinated CFRs and myelinated HTARs, may play an important role in the pathophysiological process of acute lung injury-induced respiratory responses.

A conductive pathway generated from fragments of the human red cell anion exchanger AE1

Mark D. Parker, Mark T. Young, Christopher M. Daly, Robert W. Meech, Walter F. Boron, Michael J. A. Tanner (2007) Department of 1Biochemistry and 2Physiology, University of Bristol,The Journal of Physiology 581 (1), 33–50. doi:10.1113/jphysiol.2007.128389
Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl–HCO3 exchanger with 12–14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive 36Cl-influx pathway, reminiscent of intact AE1. In the present study, they create a single construct, AE1Δ(6: 7), representing the intact protein lacking TMs 6 and 7. They expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. They found that, whereas AE1Δ(6: 7) has some electroneutral Cl–base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys539Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Δ(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3 ≈ I > NO2 > Br > Cl > SO42 ≈ HCO3 ≈ gluconate ≈ aspartate ≈ cyclamate . It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl–HCO3 exchangers, trout AE1 and mammalian SLC26A7.

Excitability of human motor cortex inputs prior to grasp

Gita Prabhu, Martin Voss, Thomas Brochier, Luigi Cattaneo, Patrick Haggard, Roger Lemon (2007) Excitability of human motor cortex inputs prior to grasp The Journal of Physiology 581 (1), 189–201. doi:10.1113/jphysiol.2006.123356Issue online:04 May 2007
Transcranial magnetic stimulation (TMS) was used to investigate corticospinal excitability during the preparation period preceding visually guided self-paced grasping. While subjects prepare to grasp a visible object, paired-pulse TMS at a specific interval facilitates motor-evoked potentials (MEPs) in hand muscles in a manner that varies with the role of the muscle in shaping the hand for the upcoming grasp. This anticipatory modulation may reflect transmission of inputs to human primary motor cortex (M1) for visuomotor guidance of hand shape. Conversely, single-pulse TMS is known to suppress MEPs during movement preparation. They investigate the time course of single- and paired-pulse MEP modulation. TMS was delivered over M1, at different time intervals after visual presentation of either a handle or a disc to healthy subjects. Participants were instructed to view the object, and later to grasp it when given a cue. During grasp there was a specific pattern of hand muscle activity according to the object grasped. MEPs were evoked in these muscles by TMS delivered prior to grasp. Paired-pulse MEPs were facilitated, whilst single-pulse MEPs were suppressed. The pattern of facilitation matched the object-specific pattern of muscle activity for TMS pulses delivered 150 ms or more after object presentation. However, this effect was not present when TMS was delivered immediately after object presentation, or if the delivery of TMS was given separately from the cue to perform the grasp action. These results suggest that object-related information for preparation of appropriate hand shapes reaches M1 only immediately preceding execution of the grasp.

Vesicular ATP Is the Predominant Cause of Intercellular Calcium Waves in Astrocytes

Published online 15 May 2007David N. Bowser and Baljit S. KhakhMedical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, UK
Brain astrocytes signal to each other and neurons. They use changes in their intracellular calcium levels to trigger release of transmitters into the extracellular space. These can then activate receptors on other nearby astrocytes and trigger a propagated calcium wave that can travel several hundred micrometers over a timescale of seconds. A role for endogenous ATP in calcium wave propagation in hippocampal astrocytes has been suggested, but the mechanisms remain incompletely understood. They explored how calcium waves arise and directly tested whether endogenously released ATP contributes to astrocyte calcium wave propagation in hippocampal astrocytes. They find that vesicular ATP is the major, if not the sole, determinant of astrocyte calcium wave propagation over distances between 100 and 250 µm, and 15 s from the point of wave initiation. These actions of ATP are mediated by P2Y1 receptors. In contrast, metabotropic glutamate receptors and gap junctions do not contribute significantly to calcium wave propagation. Data suggest that endogenous extracellular astrocytic ATP can signal over broad spatiotemporal scales.

Neurovascular coupling in the mammalian brain

Experimental Physiology 92.4 pp 641-646DOI: 10.1113/expphysiol.2006.036368Jessica A. Filosa1 and Víctor M. Blanco11 Department of Psychiatry, University of Cincinnati, 2170 East Galbraith Road, Room 239-A, Cincinnati, OH 45237, USA
Normal brain function requires proper supply of oxygen and glucose in a timely and local manner. This is achieved through an orchestrated intercellular communication between neurones, astrocytes and microvessels that results in a rapid and restricted increase in cerebral blood flow, a process known as neurovascular coupling. Astrocytic end-feet make close contacts with neuronal synapses and blood vessels and, given their ability to release vasoactive signals following neuronal activation, have been recognized as key intermediaries in the neurovascular response. Both dilating and constricting signals appear to be released from astrocytes upon increases in intracellular Ca2+ concentration, and both dilatation and constriction of brain vessels have been observed in previous studiesthe rise in astrocytic Ca2+ following neuronal activation leads not only to the rapid activation of calcium-activated K+ channels in astrocytic end-feet, but also to their modulation by metabolites of the arachidonic acid pathway, which in general have been proposed to act on vascular smooth muscle cells rather than on astrocytes. They propose that this latter mechanism may in turn modulate K+ signalling from astrocytes to smooth muscle cells, influencing the overall effects of the vasodilating and vasoconstricting signals released during neuronal activation.

O2 sensing at the mammalian carotid body: why multiple O2 sensors and multiple transmitters?

Experimental Physiology 91.1 pp 17-23DOI: 10.1113/expphysiol.2005.031922Nanduri R Prabhakar11 Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 440109, USA
Carotid bodies are the sensory organs for detecting systemic hypoxia and the ensuing reflexes prevent the development of tissue/cellular hypoxia. Although every mammalian cell responds to hypoxia, O2 sensing by the carotid body is unique in that it responds instantaneously (within seconds) to even a modest drop in arterial PO2. Sensing hypoxia in the carotid body requires an initial transduction step involving O2 sensor(s) and transmitter(s) for subsequent activation of the afferent nerve endingThe currently proposed O2 sensors include various haem-containing proteins, and a variety of O2-sensitive K+ channels. It is proposed that the transduction involves an ensemble of, and interactions between, haem-containing proteins and O2-sensitive K+-channel proteins functioning as a ‘chemosome’; the former for conferring sensitivity to wide range of PO2 values and the latter for the rapidity of the response. Hypoxia releases both excitatory and inhibitory transmitters from the carotid body.ATP is emerging as an important excitatory transmitter for afferent nerve activation by hypoxia. Whereas the inhibitory messengers act in concert with excitatory transmitters like a ‘push–pull’ mechanism to prevent over excitation, conferring the ‘slowly adapting’ nature of the afferent nerve activation during prolonged hypoxia

Interaction between genioglossus and diaphragm responses to transcranial magnetic stimulation in awake humans

Experimental Physiology 92.4 pp 739-747DOI: 10.1113/expphysiol.2007.037572Wei Wang1,2, Thomas Similowski3,4 and Frédéric Sériès1,4
The modulation of activity of the upper airway dilator and respiratory muscles plays a key role in the regulation of ventilation, but little is known about the link between their neuromuscular activation processes in vivo. This study investigated genioglossus and diaphragm responses to transcranial magnetic stimulation applied in different facilitatory conditions. The amplitude and latency of motor-evoked potential responses and the stimulation intensity threshold leading to a motor response (motor threshold) were recorded with stimulation applied at the vertex and anterolateral area in 13 awake normal subjects. Stimuli were applied during inspiration with and without resistance, during expiration with and without maximal tongue protrusion and during deep inspiration. In each stimulation location and condition, no diaphragmatic response was obtained without previous genioglossus activity (diaphragmatic and genioglossus responses latencies during expiration: 18.1 ± 2.9 and 6.3 ± 2.6 ms, respectively, mean ± S.D., P < 0.01). Genioglossus motor-evoked potential amplitude, latency and motor threshold were significantly modified with tongue protrusion with a maximal effect observed for stimulation in the anterolateral area. Deep inspiration was associated with a significant facilitatory effect on both genioglossus and diaphragm motor responses. The facilitatory effects of respiratory and non-respiratory manoeuvres were also observed during focal stimulation where isolated genioglossus responses were observed. Genioglossus and diaphragm differed in their motor threshold both at baseline and following facilitatory manoeuvres. Conclusions: (1) transcranial magnetic stimulation-induced genioglossus response systematically precedes that of diaphragm; (2) this sequence of activation is not modified by respiratory and non-respiratory manoeuvres; and (3) the genioglossus and diaphragm are differently influenced by these manoeuvres in terms of latency of the motor response and of motor threshold.

An excitatory connection between the DMH and PVN that uses tachykinin NK1 receptors

The dorsomedial hypothalamus (DMH) innervates the paraventricular nucleus (PVN) with substance P (SP) immunoreactive neurones. The PVN itself powerfully influences both the neuroendocrine and the cardiovascular systems.Glutamate application to the DMH increased action current frequency in the PVN. This effect was prevented by the glutamate antagonist kynurenic acid or by synaptic block with a high-Mg2+ low-Ca2+ buffer solution.the selective tachykinin NK1 receptor antagonist L-703606 also inhibited DMH-to-PVN neurotransmission.an excitatory connection between the DMH and PVN that uses tachykinin NK1 receptors. This pathway may be important for the hypothalamic control of neuroendocrine and/or cardiovascular function

Reference: Experimental Physiology 92.4 pp 671-676DOI: 10.1113/expphysiol.2007.037457 Matthew D. Womack1 and Richard Barrett-Jolley1Department of Veterinary Preclinical Sciences, Veterinary Sciences Building, Brownlow Hill & Crown Street, University of Liverpool, Liverpool L69 7ZJ, UK