Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin

The scorpion toxin peptide BeKm-1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether-a-go-go-related gene potassium current (I(HERG)) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I(HERG) by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use- and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.     

Effect of protein kinase A-mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein

Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells. Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease. Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function. However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear. In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385-550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers. PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric. This monomeric state was also produced at the lower PKA concentration and physiological ionic strength. Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy. The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo.     

Stimulated shedding of vascular cell adhesion molecule 1 (VCAM-1) is mediated by tumor necrosis factor-alpha-converting enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-alpha-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.     

Threshold effects of respiratory muscle work on limb vascular resistance

The purpose of this study was to determine whether the human diaphragm, like limb muscle, has a threshold of force output at which a metaboreflex is activated causing systemic vasoconstriction. We used Doppler ultrasound techniques to quantify leg blood flow (Q(L)) and utilized the changes in mouth twitch pressure (DeltaP(M)T) in response to bilateral phrenic nerve stimulation to quantify the onset of diaphragm fatigue. Six healthy male subjects performed four randomly assigned trials of identical duration (8 +/- 2 min) and breathing pattern [20 breaths/min and time spent on inspiration during the duty cycle (time spent on inspiration/total time of one breathing cycle) was 0.4] during which they inspired primarily with the diaphragm. For trials 1-3, inspiratory resistance and effort was gradually increased [30, 40, and 50% maximal inspiratory pressure (MIP)], diaphragm fatigue did not occur, and Q(L), limb vascular resistance (LVR), and mean arterial pressure remained unchanged from control (P > 0.05). The fourth trial utilized the same breathing pattern with 60% MIP and caused diaphragm fatigue, as shown by a 30 +/- 12% reduction in P(M)T with bilateral phrenic nerve stimulation. During the fatigue trial, Q(L) and LVR were unchanged from baseline at minute 1, but LVR rose 36% and Q(L) fell 25% at minute 2 and by 52% and 30%, respectively, during the final minutes of the trial. Both LVR and Q(L) returned to control within 30 s of recovery. In summary, voluntary increases in inspiratory muscle effort, in the absence of fatigue, had no effect on LVR and Q(L), whereas fatiguing the diaphragm elicited time-dependent increases in LVR and decreases in Q(L). We attribute the limb vasoconstriction to a metaboreflex originating in the diaphragm, which reaches its threshold for activation during fatiguing contractions.     

The essential role of carotid body chemoreceptors in sleep apnea

Sleep apnea is attributable, in part, to an unstable ventilatory control system and specifically to a narrowed "CO2 reserve" (i.e., the difference in P(a)CO2 between eupnea and the apneic threshold). Findings from sleeping animal preparations with denervated carotid chemoreceptors or vascularly isolated, perfused carotid chemoreceptors demonstrate the critical importance of peripheral chemoreceptors to the ventilatory responses to dynamic changes in P(a)CO2. Specifically, (i) carotid body denervation prevented the apnea and periodic breathing that normally follow transient ventilatory overshoots; (ii) the CO2 reserve for peripheral chemoreceptors was about one half that for brain chemoreceptors; and (iii) hypocapnia isolated to the carotid chemoreceptors caused hypoventilation that persisted over time despite a concomitant, progressive brain respiratory acidosis. Observations in both humans and animals are cited to demonstrate the marked plasticity of the CO2 reserve and, therefore, the propensity for apneas and periodic breathing, in response to changing background ventilatory stimuli.     

Protein kinase C-epsilon-null mice have decreased hypoxic pulmonary vasoconstriction

PKC contributes to regulation of pulmonary vascular reactivity in response to hypoxia. The role of individual PKC isozymes is less clear. We used a knockout (null, -/-) mouse to test the hypothesis that PKC-epsilon is important in acute hypoxic pulmonary vasoconstriction (HPV). We asked whether deletion of PKC-epsilon would decrease acute HPV in adult C57BL6xSV129 mice. In isolated, salt solution-perfused lung, reactivity to acute hypoxic challenges (0% and 3% O(2)) was compared with responses to angiotensin II (ANG II) and KCl. PKC-epsilon -/- mice had decreased HPV, whereas responses to ANG II and KCl were preserved. Inhibition of nitric oxide synthase (NOS) with nitro-l-arginine augmented HPV in PKC-epsilon +/+ but not -/- mice. Inhibition of Ca(2+)-gated K(+) channels (K(Ca)) with charybdotoxin and apamin did not enhance HPV in -/- mice relative to wild-type (+/+) controls. In contrast, the voltage-gated K(+) channel (K(V)) antagonist 4-aminopyridine increased the response of -/- mice beyond that of +/+ mice. This suggested that increased K(V) channel expression could contribute to blunted HPV in PKC-epsilon -/- mice. Therefore, expression of the O(2)-sensitive K(V) channel subunit Kv3.1b (100-kDa glycosylated form and 70-kDa core protein) was compared in whole lung and pulmonary artery smooth muscle cell (PASMC) lysates from +/+ and -/- mice. A subtle increase in Kv3.1b was detected in -/- vs. +/+ whole lung lysates. A much greater rise in Kv3.1b expression was found in -/- vs. +/+ PASMC. Thus deletion of PKC-epsilon blunts murine HPV. The decreased response could not be attributed to a general loss in vasoreactivity or derangements in NOS or K(Ca) channel activity. Instead, the absence of PKC-epsilon allows increased expression of K(V) channels (like Kv3.1b) to occur in PASMC, which likely contributes to decreased HPV.     

Three novel Bid proteins generated by alternative splicing of the human Bid gene

Bid, a BH3-only Bcl-2 protein, is activated by proteolytic cleavage exposing the BH3 domain, which then induces apoptosis by interacting with pro-apoptotic Bcl-2 family proteins (e.g. Bax and Bak) at the mitochondrial surface. The arrangement of domains within Bid suggested that Bid function might be regulated in part by alternative splicing. We have determined the gene structure of human Bid and identified a number of novel exons. We have also demonstrated endogenous mRNA and protein expression for three novel isoforms of Bid, generated using these exons. Bid(S) contains the N-terminal regulatory domains of Bid without the BH3 domain; Bid(EL) corresponds to full-length Bid with additional N-terminal sequence; and Bid(ES) contains only the Bid sequence downstream of the BH3 domain. Expression of these isoforms is regulated during granulocyte maturation. In functional studies Bid(EL) induces apoptosis, whereas Bid(S) abrogates the pro-apoptotic effects of truncated Bid and inhibits Fas-mediated apoptosis. Bid(ES) induces apoptosis but is also able to partially inhibit the pro-apoptotic effects of truncated Bid. These three novel endogenously expressed isoforms of Bid are distinct in their expression, their cellular localization, and their effects upon cellular apoptosis. Differential expression of these novel Bid isoforms may regulate the function of Bid following cleavage and thus influence the fate of cells exposed to a range of pro-apoptotic stimuli.     

Increased propensity for apnea via dopamine-induced carotid body inhibition in sleeping dogs.

We determined the effects of specific carotid body chemoreceptor inhibition on the propensity for apnea during sleep. We reduced the responsiveness of the carotid body chemoreceptors using intravenous dopamine infusions during non-rapid eye movement sleep in six dogs. Then we quantified the difference in end-tidal Pco(2) (Pet(CO(2))) between eupnea and the apneic threshold, the "CO(2) reserve," by gradually reducing Pet(CO(2)) transiently with pressure support ventilation at progressively increased tidal volume until apnea occurred. Dopamine infusions decreased steady-state eupneic ventilation by 15 +/- 6%, causing a mean CO(2) retention of 3.9 +/- 1.9 mmHg and a brief period of ventilatory instability. The apneic threshold Pet(CO(2)) rose 5.1 +/- 1.9 Torr; thus the CO(2) reserve was narrowed from -3.9 +/- 0.62 Torr in control to -2.7 +/- 0.78 Torr with dopamine. This decrease in the CO(2) reserve with dopamine resulted solely from the 20.5 +/- 11.3% increase in plant gain; the slope of the ventilatory response to CO(2) below eupnea was unchanged from normal. We conclude that specific carotid chemoreceptor inhibition with dopamine increases the propensity for apnea during sleep by narrowing the CO(2) reserve below eupnea. This narrowing is due solely to an increase in plant gain as the slope of the ventilatory response to CO(2) below eupnea was unchanged from normal control. These findings have implications for the role of chemoreceptor inhibition/stimulation in the genesis of apnea and breathing periodicity during sleep.     

A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXC chemokine ligand 16

CXC chemokine ligand (CXCL)16 and scavenger receptor for phosphatidylserine and oxidized low-density lipoprotein were independently identified as a chemokine and a scavenger receptor, respectively, but have since been shown to be identical. CXCL16 is synthesized as a transmembrane protein with its chemokine domain at the end of a mucin-rich stalk. When expressed at the cell surface, CXCL16 functions as a scavenger receptor, binding and internalizing oxidized low-density lipoprotein and bacteria. As a soluble form, CXCL16 is a chemoattractant for activated CD4+ and CD8+ T cells through binding its receptor, CXCR6. In this study, we examined the mechanisms that regulate the conversion between these two functionally distinct forms of CXCL16. We demonstrate that murine CXCL16 is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it undergoes metalloproteinase-dependent cleavage, causing the release of a fragment that constitutes the majority of the CXCL16 extracellular domain. Using a novel retroviral system for the generation of short interfering RNAs, we show that knockdown of a disintegrin and metalloproteinase (ADAM) family protease ADAM10 decreases this constitutive shedding of CXCL16. Furthermore, we show that overexpression of ADAM10 increases CXCL16 shedding, whereas overexpression of a dominant-negative form of ADAM10 lowers shedding of CXCL16 in a similar manner to short interfering RNAs. Through the modulation of ADAM10 function, we demonstrate that ADAM10-mediated constitutive shedding is a key regulator of CXCL16 cell surface expression. The identification of ADAM10 as a major protease responsible for the conversion of CXCL16 from a membrane-bound scavenger receptor to a soluble chemoattractant will provide new information for understanding the physiological function of this molecule.     

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1)

Fractalkine (CX3CL1) is an unusual member of the chemokine family that is synthesized with its chemokine domain at the end of a mucin-rich, transmembrane stalk. This membrane-bound localization allows fractalkine to function as an adhesion molecule for cells bearing its receptor, CX3CR1. In addition, fractalkine can be proteolytically released from the cell surface, generating a soluble molecule that functions as a chemoattractant similar to the other members of the chemokine family. In this study, we have examined the mechanisms that regulate the conversion between these two functionally distinct forms of fractalkine. We demonstrate that under normal conditions fractalkine is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it becomes a target for metalloproteinase-dependent cleavage that causes the release of a fragment containing the majority of the fractalkine extracellular domain. We show that the cleavage of fractalkine can be markedly enhanced by stimulating cells with phorbol 12-myristate 13-acetate (PMA), and we identify tumor necrosis factor-alpha converting enzyme (TACE; ADAM17) as the protease responsible for this PMA-induced fractalkine release. In addition, we provide data showing that TACE-mediated fractalkine cleavage occurs at a site distinct from the dibasic juxtamembrane motif that had been suggested previously based on protein sequence homologies. The identification of TACE as a major protease responsible for the conversion of fractalkine from a membrane-bound adhesion molecule to a soluble chemoattractant will provide new information for understanding the physiological function of this chemokine.