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Tight regulation of the plasma membrane proton pump ATPase (H+-ATPase) is necessary for controlling the membrane potential that energizes secondary transporters. This regulation relies on the phosphorylation of the H+-ATPase penultimate residue, a theonine, and the subsequent binding of regulatory 14-3-3 proteins, which results in enzyme activation. Using phospho-specific antibodies directed against the phosphorylable Thr of either PMA2 (Plasma membrane H+-ATPase from N. plumbaginifolia) or PMA4, we showed that the kinetics and extent of phosphorylation differ between both isoforms according to the growth or environmental conditions like cold stress.1Here, we used phospho-specific antibodies to follow PMA2 Thr phosphorylation upon acidification of the cytosol by incubating N. tabacumBY2 cells with four different weak organic acids. Increased PMA2 phosphorylation was observed for three of them, thus highlighting the role of the H+-ATPase in cell pH homeostasis.

We have isolated a novel gene (GLELO) from Mortierella alpina and its homologue (CEELO1) from Caenorhabditis elegans and demonstrate the involvement of their encoded proteins in the elongation of C18 polyunsaturated fatty acids.

1. The rates of entry of noradrenaline to plasma and of removal of noradrenaline from plasma, and plasma noradrenaline concentration, were determined in normal subjects and in patients with essential hypertension. Neuronal uptake of noradrenaline was assessed from the plasma tritiated noradrenaline disappearance curve, after infusion to steady state. 2. Noradrenaline disappearance was biexponential. Rapid removal was dependent on neuronal uptake, being slowed if neuronal noradrenaline uptake was reduced, either by desipramine in normal subjects, or in patients with sympathetic nerve dysfunction (autonomic insufficiency). 3. In 10 of 41 hypertensive patients the t1 1/2 similarly was prolonged, presumptive evidence of a defect in neuronal noradrenaline uptake. Endogenous noradrenaline escaping uptake after release, and spilling over into plasma, and plasma noradrenaline concentration, were increased in these patients. 4. Defective neuronal uptake of noradrenaline, by exposing adrenoreceptors to high local transmitter concentration, may be important in the pathogenesis of essential hypertension in some patients.

1. The influence of age on the rate of spillover of noradrenaline into plasma, clearance of noradrenaline from plasma, and plasma noradrenaline concentration at rest was studied in 34 healthy subjects aged 20–69 years. 2. The plasma concentration of noradrenaline was dependent on age, values being higher in older subjects. 3. This age-dependence of plasma noradrenaline concentration was due principally to a reduced clearance of noradrenaline from the circulation in older subjects. 4. The rate of spillover of noradrenaline into plasma was little influenced by age. The higher plasma noradrenaline values found in older subjects do not seem to be due to an increase in sympathetic nervous system tone with aging.

The univalent cations Na+, K+and H+are known to play a critical role in regulating growth in a number of mammalian cell types. In this study we used tissue culture to explore the ways in which two cation transport processes, known to affect the intracellular concentrations of these ions, and Na-H exchange and the Na-K pump, affect serum proliferation of rat aortic smooth muscle. Exposure of quiescent serum-deprived smooth muscle to 10 serum increased both the Na-H exchange and the activity of the Na-K pump. This activation of Na-H exchange increased intracellular pH in proliferating cells by 0.22 units. Attenuation of exchange activity by 40 μmol/l ethylisopropylamiloride (EIPA) reduced the smooth muscle replication rate by 79. Attenuation of the Na-K pump activity also reduced the replication rate. These results indicate that alterations in the activities of either Na-H exchange or Na-K pump activity will influence smooth muscle cell replication.

A study was made of 187 patients suffering from overdosage by means of clinical records and drug analysis. Drugs were detected in 152 patients. The groups of drugs most frequently involved in these patients were sedatives (51$md7%), tricyclic antidepressants (21$md8%) and analgesics (11$md2%). This overall pattern differed little from that determined by clinical diagnosis. However, discrepancies between clinical diagnosis and laboratory analysis were found in 43 patients. A retrospective assessment was made from clinical records of the influence of drug assay results upon clinical management Decisions as to the need for admission to hospital and decisions relating to the management of patients suffering from overdoses of analgesics and tricyclic antidepressants were aided by drug analysis.

The effects of a single dose and of two equally divided doses of timolol were compared in a double‐blind trial in 15 patients with essential hypertension. The amount of timolol per day remained constant for each patient, but individual patients received different amounts (from 10 mg to 30 mg/day). Timolol combined with a diuretic produced similar lowering of blood pressure in patients who required 10 mg, 20 mg or 30 mg of timolol per day when given either as one dose or in two equal doses. Plasma concentrations of timolol were high enough to be a major contributing factor to the long duration of the hypotensive response.

The pinceau is a cerebellar structure formed by GABA-ergic descending basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although it exerts a powerful inhibitory influence on the output of the cerebellar cortex, the exact ultrastructural organization and function of this structure are not understood. The unique morphology of the pinceau lies in several elements: lack of synaptic contacts between basket cell terminals and the initial axonal segment, the absence of postsynaptic densities, and heavy ensheathment of the initial segment with glial processes, making astrocytes the only intervening cellular element between the two types of axonal structures. Several plasticity related proteins in this area have been previously characterized by immunocytochemistry. Those include nitric oxide synthase, PSD95, GABA transporters, and several voltage gated potassium channel subunits. We are using a combination of confocal and electron microscopy, correlated photooxidation, EM and tomography to reveal the ultrastructural distribution of novel proteins and their relationships to morphological compartments in the pinceau.We describe here Kv3.2, a recently discovered member of the Shaw-like subfamily of potassium channels, and the correlated distribution of other proteins described below. Pre-embedding immunolabeling, electron microscopy and tomography showed that Kv3.2 was selectively localized to basket cell axons.

A scheme for drug identification in overdosed patients has been developed, using a combination of the techniques of extraction, thin layer and gas chromatography, ultra violet spectroscopy and colorimetry. 5ml plasma and 50ml urine or gastric aspirate is required for the full drug screen, which can be completed within 2h. Fifty-three drugs are presently encompassed by the screen, and include analgesics, alcohols, antihistamines, anticonvulsants, barbiturates and related compounds, narcotics, phenothiazines, sedatives, tranquillizers and tricyclic antidepressants.

The article focuses on a 2013 Reuters article which discussed the role that super foods can play in weight loss and over all health, discussed ten super foods and reported that if the U.S. population continues to partake in its unhealthy lifestyle, half of all U.S. adults will be considered obese by the year 2030.

Despite promising pre-clinical results, administration of granulocyte-colony stimulating factor (G-CSF) for improving heart function in humans after acute myocardial infarction has been disappointing. Therefore, a better understanding of the mechanism(s) of heart repair by G-CSF is urgently required. To address the potential mechanism of G-CSF, we have generated chimeric mice using G-CSF receptor knockout mice to distinguish the hematopoietic benefits of G-CSF from the direct effects of G-CSF on cardiomyocytes and blood vessels. Lethally irradiated wild-type mice were reconstituted with G-CSFR −/− bone marrow (BM) cells to generate G-CSFR BM −/− chimeras. Alternatively, lethally irradiated G-CSFR −/− mice were reconstituted with wild-type BM cells to generate G-CSFR cardiac −/− chimeras. Myocardial ischemic injury was induced by transient (60 minutes) occlusion of the left coronary artery. Mice were treated daily with saline or G-CSF for 5 days beginning 4 hours after ischemic injury. Mice were sacrificed 28 days post-injury for assessment of cardiac function and cellular repair. As previously reported in wild-type mice, G-CSFR −/− cardiac chimeras had 30–50% improved cardiac function compared with saline-treated G-CSFR −/− cardiac chimeras (ANOVA; P<0.05). This suggests that the benefits of G-CSF are NOT due to direct effects on cardiomyocytes or blood vessels, as previously proposed by Harada et al. (Nature Medicine2005; 11: 305–311). In contrast, G-CSF BM −/− chimeras had dramatically impaired cardiac repair following ischaemic injury. The most striking finding was that saline-treated G-CSFR BM −/− mice had 20% worse cardiac function and cellular repair than saline-treated control G-CSFR BM +/+ mice (ANOVA; P<0.05). Furthermore, G-CSF treatment in G-CSFR BM −/− mice produced no significant benefit on cardiac function or cellular repair. The lack of benefit of G-CSF in these mice was due to an inability of G-CSF to mobilise hematopoietic progenitors because treatment of G-CSFR BM −/− mice with the CXCR4 antagonist, AMD3100 improved cardiac function and cellular repair as well as G-CSF in control G-CSFR BM +/+ mice. Together, these results suggest that mobilization of hematopoietic progenitors is a critical component of not only the therapeutic but also physiologic effects of G-CSF for repair after acute myocardial ischemic injury.

Mechanical strain has been shown to induce mitogenesis in a rat neonatal vascular smooth muscle (VSM) cell line in a response mediated predominantly by transcription, expression, and release of platelet-derived growth factor (PDGF). We examined the effect of cyclic mechanical strain and growth factor production on mitogenic response in ovine coronary artery smooth muscle cells. Vascular smooth muscle cells were cultured from explants of left anterior descending (LAD) coronary arteries from young sheep. Cells for experiments were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. Tritiated thymidine incorporation was used as a measure of DNA synthesis. Cell membrane damage was assessed with differentially permeable nuclear staining dyes. We observed an increase in tritiated thymidine incorporation in response to strain with a temporal response identical to that observed in response to exogenous growth factors (PDGF-BB and basic fibroblast growth factor [bFGF]). Supernatant medium obtained from stretched cells induced a twofold increase in DNA synthesis in unstretched cells. The mitogenic response was abolished by monoclonal antibodies to bFGF, but not by antibodies to PDGF-AB. Studies of fluorescent dye exclusion indicated the stretching protocol caused no cell membrane damage. Thus, mechanical strain is an important stimulus for growth factor release in coronary VSM cells. The mitogenic response is mediated by release of bFGF. Am J Hypertens 2001;14:1128–1134 © 2001 American Journal of Hypertension, Ltd.

Mechanical strain has been shown to induce proliferation in neonatal vascular smooth muscle (VSM) cells. We compared the effects of strain on cultured VSM cells from epicardial and resistance coronary arteries from adult sheep hearts. Cells were grown on wells with silicone-elastomer bottoms, and subjected to strain (60 cycles/min) using a vacuum actuated strain device. 3H-thymidine incorporation was used as a measure of DNA synthesis. Strain induced increase in DNA synthesis was 1.9 fold higher in cells obtained from resistance arteries, compared to cells obtained from the left anterior descending (LAD) coronary artery. To gain further insights into the effects of strain on coronary artery VSM cells, we studied the time course of the strain-induced increase in DNA synthesis in cultured VSM cells from the LAD. We observed an increase in 3H-thymidine incorporation in response to strain with a peak at 18 hours, identical to the time to peak effect obtained in response to exogenous growth factors (PDGF and basic FGF), consistent with the continual rapid release of cellular growth factors from strained cells. Supernatant medium obtained from these cells induced a 2.01 fold increase in DNA synthesis in unstretched coronary VSM cells. Since TGF-βand basic FGF are both reported to be present in VSM cells, we explored the potential role of these factors in the strain induced response. Supernatant from LAD cells induced an inhibitory response in mink lung epithelial cells, suggesting the presence of TGF-βin the medium. In cultured LAD cells that had undergone multiple replications, the proliferative response was abolished by monoclonal antibodies to basic FGF. We conclude that mechanical strain is an important stimulus to proliferation in coronary VSM cells derived from both epicardial and resistance coronary arteries. In LAD cells, the increase in DNA synthesis may be mediated via release of autocrine growth factors, specifically TGF-βand/or basic FGF.

A gas liquid chromatographic procedure for determining therapeutic concentrations of timolol in plasma has been developed. Timolol together with an internal standard desmethyltimolol is extracted fronl 4ml of alkalinized plasma into 5% isoamyl alcohol-heptane. The compounds are back-extracted into 0.1N hydrochloric acid. The acid phase is adjusted to alkaline pH and extracted with methylene chloride: ether (1: 5). The resultant residue from evaporation of the organic phase is derivatized with heptafluorobutyrylimidazole in ethyl acetate solution to form diheptafluorobutyrate derivatives of timolol and desmethyltimolol. Quantitation is carried out by chromatography on 1% OV-17 (support Gas Chrom Q) and a gas chromatograph equipped with an electron capture detector. Detector response is linear up to a plasma timolol concentration of 125ng/ml. At 12.5ng/lnl the coefficient of variation is 12%. The limit of sensitivity is approximately 4ng/ml. The method is sufficiently accurate for pharmacokinetic analysis.

Objectives: The aim was to determine the mechanisms, particularly bicarbonate dependent mechanisms, of intracellular pH (pH_i) recovery from various acidoses in vascular smooth muscle and to explore the ATP dependency of the respective mechanisms. Methods: Experiments were conducted in rat aortic smooth muscle cells grown in primary culture and synchronised in a non-growing state by serum deprivation. pH_i was measured in cells loaded with the pH sensitive fluorescent dye, 2,7-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF). Chloride efflux was studied by determination of the rate of efflux of 36Cl over 5 min. Cells were ATP depleted by substitution of glucose in the medium by 2-deoxyglucose. Acidoses were induced by CO₂ influx and NH₃ efflux techniques. Results: In the absence of HCO₃–, the 5-(N-ethyl-N-isopropyl) amiloride (EIPA) sensitive Na+/H+ exchange accounted for the recovery from intracellular acidosis. In the presence of HCO₃– ions the response to respiratory acidosis (CO₂influx) was predominantly via activation of Na+/H+ exchange and an EIPA sensitive Na+ and HCO₃– dependent mechanism. A 4-acetamido-4-isothiocyanostilbene-2,2-sulphonic acids (SITS) sensitive Na+ dependent /medium/29-2-239-si1.gif" ALT="Formula "<$INLINEFIG_ALIGN>> mechanism which is also sensitive to EIPA makes a small contribution during severe intracellular acidosis. Under such conditions HCO₃– dependent mechanisms contributed about 40% to the overall pH, regulating capacity of vascular smooth muscle cells. However, under conditions which deplete cellular ATP these pH_i regulating mechanisms account for virtually all of theses cells ability to regulate pH_i. The inability of Na+/H+ exchange to participate in pH_i recovery under these circumstances, reduces the ability of vascular smooth muscle cells to recover pH_i, by approximately 50–60%. Chloride efflux was approximately linear over 5 min and was increased by 36% in the presence of extracellular HCO₃–. Efflux in the presence of HCO₃– was inhibited similarly by both SITS and EIPA. Conclusions: At least three transporters contribute to recovery from acidosis in vascular smooth muscle: Na+/H+ exchange, an Na+-HCO₃– cotransporter which is sensitive to EIPA, and an Na+ dependent /medium/29-2-239-si2.gif" ALT="Formula "<$INLINEFIG_ALIGN>> exchange sensitive to both SITS and EIPA. The Na+-HCO₃– cotransporter appears to be similar to that described in human vascular smooth muscle. When the Na+/H+- exchanger is attenuated by cellular ATP depletion, the alternative pathways, particularly the Na+- HCO₃– cotransporter, ensure that substantial pH_i regulatory capacity is maintained.

Intrinsic differences between vascular smooth muscle cells (VSMC) in normotension and genetic hypertension may account for the exaggerated growth response often observed in the hypertensive vasculature. To test this hypothesis, in this study we compared the vascular growth response of the spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) following induction of one kidney, one clip (1K1C) renal hypertension. SHR and WKY rats were uninephrectomized and renal hypertension (RH) induced using silver clips of 0.22 and 0.24 mm width. Four weeks later, vessel and VSMC growth were assessed in small mesenteric arteries. The systolic blood pressure (SBP) in the RH animals was significantly higher than in uninephrectomised controls, in RH-SHR with a 0.24 mm clip SBP averaged 215 ± 4 mm Hg and in RH-WKY with a 0.22 or 0.24 mm clip the SBPs averaged 214 ± 5 mm Hg and 190 ± 2 mm Hg, respectively. For the same SBP, there were no differences in medial cross-sectional areas of the small mesenteric arteries between RH-SHR and RH0.22-WKY, which averaged 1.73 ± 0.19 × 104 µm2 and 1.66 ± 0.15 × 104 µm2, respectively. Likewise, the number of VSMCs (within a precise anatomical site of the mesenteric vasculature) were not different between the RH-SHR and the RH0.22-WKY with VSMC number 7.6 ± 0.8 × 104 cells and 6.9 ± 0.4 × 104 cells, respectively. In the RH0.22-WKY vascular growth responses were generally unchanged compared with the RH0.24-WKY except for a further increase in the incidence of polyploid cells. In conclusion, the results of this study demonstrate that smooth muscle cells of the SHR are not hyperresponsive to all growth-promoting stimuli. Taken together with previous observations, it appears that sustained activity of the renin-angiotensin system may be required for exaggerated vascular growth responses in SHR. © 1997 American Journal of Hypertension, Ltd. Am J Hypertens 1997;10:43–50

We studied the hemodynamic effects of four doses of milrinone, administered by intravenous (i.v.) infusion alone and after autonomic blockade with prazosin, propranolol, atropine, and clonidine. Plasma concentrations of milrinone (50–600 ng/ml) were similar to those used for the treatment of cardiac failure and were unaltered by autonomic blockade. When given alone, milrinone induced dose-dependent increases in heart rate (maximum increase 21 ± 4, SEM, beats/min) and cardiac output (CO) (maximum 44 ± 9X) and reduced systemic vascular resistance (SVR) by a maximum of 32 ± 5X. After autonomic blockade, milrinone caused a similar fall in SVR and a smaller but significant (7 ± 2 beats/min) rise in heart rate, but no change in CO. The increase in CO produced in normal humans by acute i.v. infusions of milrinone depends on intact cardiovascular reflexes.

Cardiac hypertrophy is an adaptive response to increased wall stress in hypertension. Although the mechanisms underlying this adaptive response are not clearly understood, a genetic predisposition appears to playa role. We sought to examine the hypothesis that an inflammatory response occurs in the heart early in the course of development of hypertension. We measured blood pressure, heart weight and cardiac macrophage-monocyte invasion in 6 week old SHR and WKY rats with renal hypertension (1 Kidney-1 Clip, 1K1C) or sham-operated controls (SHAM). Macrocyte-monophage invasion was measured as the extent of immunohistochemical staining with ED1, a cytoplasmic antigen in this cell type. Measurements were made at 7 and 21 days post procedure. Renal hypertensive rats, both SHR and WKY, had higher blood pressures at both time points than SHR sham, which in turn had higher blood pressures that WKY sham rats. Heart weight/body weight ratios were highest in SHR-1 K1 C rats, followed by SHR SHAM and WKY 1K1C, and least in WKY sham rats. ED1 staining was also highest in SHR 1K-lC rats, followed by SHR sham rats, and was considerably lower in WKY rats, 1K-1C or sham. Data atthe 21 day time point are shown below:BP (mmHg)Heart wt/body wt (mg/g)ED1 (counts/HPF)SHR 1K1C2054.320.0SHR SHAM1703.415.0WKY 1K1C2033.712.5WKY SHAM1432.812.5