Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange.

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety

Activation of electroneutral K flux in Amphiuma red blood cells by N-ethylmaleimide. Distinction between K/H exchange and KCl cotransport.

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation

pH regulatory Na/H exchange by Amphiuma red blood cells.

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683-708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H+ Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events

Prehospital transdermal glyceryl trinitrate in patients with ultra-acute presumed stroke (RIGHT-2): an ambulance-based, randomised, sham-controlled, blinded, phase 3 trial

Background High blood pressure is common in acute stroke and is a predictor of poor outcome; however, large trials of lowering blood pressure have given variable results, and the management of high blood pressure in ultra-acute stroke remains unclear. We investigated whether transdermal glyceryl trinitrate (GTN; also known as nitroglycerin), a nitric oxide donor, might improve outcome when administered very early after stroke onset. Methods We did a multicentre, paramedic-delivered, ambulance-based, prospective, randomised, sham-controlled, blinded-endpoint, phase 3 trial in adults with presumed stroke within 4 h of onset, face-arm-speech-time score of 2 or 3, and systolic blood pressure 120 mm Hg or higher. Participants were randomly assigned (1:1) to receive transdermal GTN (5 mg once daily for 4 days; the GTN group) or a similar sham dressing (the sham group) in UKbased ambulances by paramedics, with treatment continued in hospital. Paramedics were unmasked to treatment, whereas participants were masked. The primary outcome was the 7-level modified Rankin Scale (mRS; a measure of functional outcome) at 90 days, assessed by central telephone follow-up with masking to treatment. Analysis was hierarchical, first in participants with a confirmed stroke or transient ischaemic attack (cohort 1), and then in all participants who were randomly assigned (intention to treat, cohort 2) according to the statistical analysis plan. This trial is registered with ISRCTN, number ISRCTN26986053. Findings Between Oct 22, 2015, and May 23, 2018, 516 paramedics from eight UK ambulance services recruited 1149 participants (n=568 in the GTN group, n=581 in the sham group). The median time to randomisation was 71 min (IQR 45–116). 597 (52%) patients had ischaemic stroke, 145 (13%) had intracerebral haemorrhage, 109 (9%) had transient ischaemic attack, and 297 (26%) had a non-stroke mimic at the final diagnosis of the index event. In the GTN group, participants’ systolic blood pressure was lowered by 5·8 mm Hg compared with the sham group (p<0·0001), and diastolic blood pressure was lowered by 2·6 mm Hg (p=0·0026) at hospital admission. We found no difference in mRS between the groups in participants with a final diagnosis of stroke or transient ischaemic stroke (cohort 1): 3 (IQR 2–5; n=420) in the GTN group versus 3 (2–5; n=408) in the sham group, adjusted common odds ratio for poor outcome 1·25 (95% CI 0·97–1·60; p=0·083); we also found no difference in mRS between all patients (cohort 2: 3 [2–5]; n=544, in the GTN group vs 3 [2–5]; n=558, in the sham group; 1·04 [0·84–1·29]; p=0·69). We found no difference in secondary outcomes, death (treatment-related deaths: 36 in the GTN group vs 23 in the sham group [p=0·091]), or serious adverse events (188 in the GTN group vs 170 in the sham group [p=0·16]) between treatment groups. Interpretation Prehospital treatment with transdermal GTN does not seem to improve functional outcome in patients with presumed stroke. It is feasible for UK paramedics to obtain consent and treat patients with stroke in the ultraacute prehospital setting

A trial for the use of qigong in the treatment of pre and mild essential hypertension: a study protocol for a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Hypertension is a risk factor for cardiovascular disease, and the prevalence of hypertension tends to increase with age. Current treatments for hypertension have side effects and poor adherence. Qigong has been studied as an alternative therapy for hypertension; however, the types of qigong used in those studies were diverse, and there have not been many well-designed randomized controlled trials.</p> <p>Our objectives are the following: 1) To evaluate the effects of qigong on blood pressure, health status and hormone levels for pre- or mild hypertension. 2) To test the methodological appropriateness of this clinical trial and calculate a sample size for future randomized trials.</p> <p>Methods</p> <p>Forty subjects with pre- or mild hypertension will be randomized to either the qigong exercise group or the non-treated group. Participants in the qigong group will conduct qigong exercises 5 times per week for 8 weeks, and participants in the non-treated group will maintain their current lifestyle, including diet and exercise. The use of antihypertensive medication is not permitted. The primary endpoint is a change in patient blood pressure. Secondary endpoints are patient health status (as measured by the SF-36 and the MYMOP2 questionnaires) and changes in hormone levels, including norepinephrine, epinephrine, and cortisol.</p> <p>Discussion</p> <p>This study will be the first randomized trial to investigate the effectiveness of qigong exercises for the treatment of pre- and mild hypertension. The results of this study will help to establish the optimal approach for the care of adults with pre- or mild hypertension.</p> <p>Trial registration</p> <p>Clinical Research Information Service KCT0000140</p

Cell entry of a host targeting protein of oomycetes requires gp96

This work is supported by the [European Community’s] Seventh Framework Programme [FP7/2007–2013] under grant agreement no. [238550] (L.L., J.D.-U., C.J.S., P.v.W.); BBSRC [BBE007120/1, BB/J018333/1 and BB/G012075/1] (F.T., I.d.B., C.J.S., S.W., P.v.W.); Newton Global Partnership Award [BB/N005058/1] (F.T., P.v.W.), the University of Aberdeen (A.D.T., T.R., C.J.S., P.v.W.) and Deutsche Forschungsgemeinschaft [CRC1093] (P.B., T.S.). We would like to acknowledge the Ministry of Higher Education Malaysia for funding INA. We would like to thank Brian Haas for his bioinformatics support. We would like to acknowledge Neil Gow and Johannes van den Boom for critical reading of the manuscript. We would like to acknowledge Svetlana Rezinciuc for technical help with pH-studies.Peer reviewedPublisher PD

Noncontrast Computed Tomography Signs as Predictors of Hematoma Expansion, Clinical Outcome, and Response to Tranexamic Acid in Acute Intracerebral Hemorrhage

Background and Purpose— Blend, black hole, island signs, and hypodensities are reported to predict hematoma expansion in acute intracerebral hemorrhage. We explored the value of these noncontrast computed tomography signs in predicting hematoma expansion and functional outcome in our cohort of intracerebral hemorrhage. Methods— The TICH-2 (Tranexamic acid for IntraCerebral Hemorrhage-2) was a prospective randomized controlled trial exploring the efficacy and safety of tranexamic acid in acute intracerebral hemorrhage. Baseline and 24-hour computed tomography scans of trial participants were analyzed. Hematoma expansion was defined as an increase in hematoma volume of >33% or >6 mL on 24-hour computed tomography. Poor functional outcome was defined as modified Rankin Scale of 4 to 6 at day 90. Multivariable logistic regression was performed to identify predictors of hematoma expansion and poor functional outcome. Results— Of 2325 patients recruited, 2077 (89.3%) had valid baseline and 24-hour scans. Five hundred seventy patients (27.4%) had hematoma expansion while 1259 patients (54.6%) had poor functional outcome. The prevalence of noncontrast computed tomography signs was blend sign, 366 (16.1%); black hole sign, 414 (18.2%); island sign, 200 (8.8%); and hypodensities, 701 (30.2%). Blend sign (adjusted odds ratio [aOR] 1.53 [95% CI, 1.16–2.03]; P=0.003), black hole (aOR, 2.03 [1.34–3.08]; P=0.001), and hypodensities (aOR, 2.06 [1.48–2.89]; P0.05). Conclusions— Blend sign, black hole sign, and hypodensities predict hematoma expansion while black hole sign, hypodensities, and island signs predict poor functional outcome. Noncontrast computed tomography signs did not predict a better response to tranexamic acid. Clinical Trial Registration— URL: https://www.isrctn.com. Unique identifier: ISRCTN93732214