The lymphocyte Na+/H+ antiport: activation in primary hypertension and during chronic NaCl-loading

Abstract. Increased activity of the Na⁺/H⁺ antiport may be a major abnormality in essential hypertension. The activity of this transport system was investigated in lymphocytes from 13 patients with untreated essential hypertension (Ht) and 13 normotensive control subjects (Nt) on an ad libitum (130–170 mmol d^-1) NaCl intake. Furthermore, the effects of different states of NaCl balance on lymphocyte Na⁺/H⁺ antiport were evaluated in two groups of Nt volunteers receiving 20 vs. 300 mmol d^-1 (n= 8) and 85 vs. 200 mmol d^-1 (n= 14) of NaCl for 1 week each and in seven Ht patients (20 vs. 300 mmol NaCl d^-1 for 1 week each). Additionally, during the 20 and 300 mmol/d NaCl intake red blood cell membrane transport was studied in eight subjects. For the determination of lymphocyte antiport activity, cells were loaded with the cytosolic pH (pH_i) indicator bis-carboxyethyl carboxyfluorescein (BCECF-AM) and acidified by addition of different amounts of Na⁺-propionate (5–40 mM). Initial pH_i-recovery was taken as the activity of the antiport system and plotted against pH_i-values after acidification. Non-linear regression analysis yielded higher ’apparent’ maximal transport rates in Ht than Nt (Nt: 2·00 pL 0·22; Ht: (3·81 pL 0·59)·10^-3 s^-1; P < 0·025). In contrast, baseline pH_i-values and pH_i-values at half-maximal activity (pK) were identical in Nt and Ht. In normotensive control subjects on an NaCl intake of 20, 85, 200 and 300 mmol d^-1 for 7 d, ’apparent’ maximal transport rates averaged 2.75 0·20, 2·89 0·17, 2·81 ± 0·18 and (3·62 ± 0·25) · 10^-3 s^-1, respectively. Thus, antiport activity was significantly (P < 0·05) stimulated on the 300 mmol d^-1 intake as compared to the three other NaCl intakes. The extreme intakes of NaCl (20 vs. 300 mmol d^-1) in normotensive volunteers did not affect the erythrocyte Na⁺/K⁺ pump, Na⁺/K⁺ cotransport and Na⁺/Li⁺ countertransport. Our study supports the concept that a group of patients with primary hypertension exhibit an activated Na⁺/H⁺ antiport. Furthermore, our data demonstrate that a chronic high intake of NaCl is associated with an increase in lymphocyte antiport activity towards the high values observed in primary hypertension.     

Acute saline infusion induces extracellular acidification and activation of the Na+/H+ exchanger in man

The effect of acute expansion of the extracellular fluid volume (ECV) with isotonic (0.9%) saline on the activity of the lymphocyte Na⁺/H⁺ antiport (NHE) was studied in a total of 18 healthy volunteers. Saline was infused at a constant rate so that 4 mmol kg^?1 b.w. was administered over 2 h. NHE activity was measured by quantifying cytosolic pH (pH_i) recovery following acidification of the cells with propionic acid and by pH clamping at various pH_i values between 7.2 and 5.8 using nigericin. Both methods demonstrate NHE activation associated with intravenous saline infusion, the kinetic difference being a marked decrease in the Hill coefficient n from 3.28 ± 0.21 (SEM) to 2.22 ± 0.11 in the absence of changes in baseline pH_i (7.14 ± 0.02 vs. 7.08 ± 0.02; P = 0.15), V_max (42.8 ± 2.7 vs. 48.1 ± 2.8 mmol L^?1 min^?1; P = 0.08) and pK (6.32 ± 0.04 vs. 6.35 ± 0.02). NHE activation was associated with significant decreases in serum chloride (P = 0.016), calcium (P = 0.008), total cholesterol (P = 0.008), low-density lipoproteins (P = 0.016) and high-density lipoproteins (P = 0.008). Moreover, saline infusion induced extracellular acidification with a decrease in pH from 7.39 ± 0.01 to 7.37 ± 0.01 (P = 0.016), HCO₃^? from 23.3 ± 0.43 mmol L^?1 to 21.3 ± 0.25 mmol L^?1 (P = 0.008) and base excess from ?1.03 ± 0.38 mmol L^?1 to ?3.00 ± 0.31 mmol L^?1 (P = 0.008). Our results show for the first time that acute ECV expansion with isotonic saline is followed by an activation of the lymphocyte NHE. The underlying mechanism(s) remain to be investigated. However, the demonstration in our study of marked changes in acid–base balance induced by acute saline points to a possible inter-relationship of antiporter activation and extracellular acidification.     

Kinetics of 13CO2 elimination after ingestion of 13C bicarbonate: the effects of exercise and acid base balance

Abstract. In order to investigate the effects of muscular work and preceding exercise on the retention of exogenous labelled bicarbonate, we studied the effects of oral administration of [¹³C]bicarbonate (0·1 mg kg^-1) in five subjects at rest before exercise and during and after 1 h of treadmill walking at 73% VO_2max on three separate occasions. Elimination of CO₂ from labelled bicarbonate was 62·6±8·1% at rest, 103·6±11·3% during exercise (P<0·01) and 43·0±4·7% during recovery from exercise (P= 0·01). During exercise mean residence time (MRT) was shorter than at rest (35±7 min vs. 54±9min, P < 0·02) and CO₂ pool size was larger (998±160 ml CO₂kg^-1, vs. 194±28ml CO₂kg^-1, P < 0·001). Compared to values obtained at rest, during recovery from exercise, MRT and CO₂ pool size were reduced (34±5min, P < 0·05; 116±19 ml CO₂kg^-1, P < 0·02, respectively). In an additional five subjects acidosis and alkalosis were induced prior to administration of oral [¹³C]bicarbonate at rest. Elimination of bicarbonate was lower during acidosis (46·1±5·6%, P < 0·01) but was unaltered (50·9±5·6%, NS) during alkalosis, compared to the values obtained at resting pH. During acidosis MRT and CO₂ pool size decreased (37±3min, P<0·01 and 123±10ml CO₂kg^-1, P < 0·01, respectively) whereas in alkalosis MRT was unchanged (65±8 min NS) but CO₂ pool size was increased (230±23ml CO₂kg^-1, P < 0·05). The kinetics of elimination of ¹³CO₂ from administered bicarbonate after exercise are different to those at rest and resemble acidosis. The appropriate correction factor for sequestered ¹³C should be used in metabolic studies of the post-exercise state when using ¹³C tracers.     

WNK4 regulates airway Na+ transport: study of familial hyperkalaemia and hypertension

Background WNK [With No K (lysine)] kinases are essential for regulation of blood pressure and potassium homeostasis. WNK4 expression was recently found not only in the distal nephron but also in chloride‐transporting epithelia. To establish a physiological role for this distribution we studied patients with familial hyperkalaemia and hypertension (FHH), [pseudohypoaldosteronism type II (PHAII)], which is caused by mutations in WNK4. Design Measurement of nasal potential difference (NPD) and sweat electrolytes were performed in controls, in six subjects with FHH and ten subjects with cystic fibrosis (CF). Results Basal NPD was higher in FHH compared with controls (n = 20): 22·8 ± 5·7 vs. 16·2 ± 5·3 mV, respectively (P = 0·014). Maximal response to amiloride was also higher in FHH compared with controls: 14·8 ± 3·5 vs. 10·0 ± 4·8 mV, respectively (P = 0·03). In CF these values were 42·9 ± 9·3 and 29·9 ± 7·4 mV, respectively. The kinetics of the amiloride effect were faster in FHH, and as first reported here also in CF, compared with controls. At 30 s, amiloride‐inhibitable residual PD in FHH was 50 ± 30 vs. 81 ± 9% in controls (P = 0·0003) and 56 ± 7% in CF. The response to chloride‐free and isoproterenol solutions, which determines chloride transport activity, was similar in FHH compared with controls [16·0 ± 8·6 vs. 10·4 ± 5·9 mV (P = 0·08)]. Sweat conductivity in FHH was 49·7 ± 7·3 vs. 38·2 ± 8·1 mmol (NaCl eq) L^?1 in 16 controls (P = 0·007) and 94·0 ± 19·3 in CF. Conclusions Mutant WNK4 increases Na⁺ transport in airways, and therefore it is regulated by wild‐type WNK4. This may be caused by a regulation of ENaC or a K⁺ channel.     

Erythrocyte Na+–H+ exchanger kinetics and Na+–Li+ countertransport activity in essential hypertensive patients

The authors measured Na⁺–H⁺ exchanger kinetics together with Na⁺–Li⁺ countertransport V _max in the erythrocytes of 21 subjects with essential hypertension and 16 normotensive control subjects. Na⁺–H⁺ exchanger V _max appeared to be increased in patients with essential hypertension, while the Na⁺–H⁺ exchanger affinity for intracellular proton sites ( K _50%) proved to be unchanged and the index of cooperativity among intracellular proton binding sites as measured by Hill's coefficient (Hill's n ) decreased as compared with normotensive control subjects. Na⁺–Li⁺ countertransport V _max appeared to be higher in patients with essential hypertension than in control subjects. The authors were unable to find any correlations between Na⁺–H⁺ exchanger kinetic parameters and metabolic variables such as parameters of insulin resistance and plasma lipids. On the basis of the data obtained, erythrocyte Na⁺–H⁺ exchanger activity was found to be abnormal in two kinetic variables in essential hypertensive patients and showed no simple linear correlations with the main variables of glucose metabolism, plasma lipids, renin or aldosterone.     

Red blood cell sodium transport in patients with cirrhosis

Patients with advanced cirrhosis have abnormal sodium homoeostasis. The study was undertaken to quantify the sodium transport across the plasma membrane of red blood cells (RBC) in patients with cirrhosis. RBC efflux and influx of sodium were studied in vitro with tracer ²²Na⁺ according to linear kinetics in 24 patients with cirrhosis and 14 healthy controls. The sodium efflux was modified by ouabain (O), furosemide (F) and a combination of O and F (O + F). RBC sodium was significantly decreased (4·6 versus control 6·3 mmol l^?1, P<0·001) and directly related to serum sodium (r = 0·57, P<0·05). The RBC fractional sodium efflux was higher in patients with cirrhosis (+46%, P<0·01) compared to controls. Inhibition in both high (145 mmol l^?1)‐ and low (120 mmol l^?1)‐sodium buffers showed that the F‐insensitive sodium efflux was twice as high in cirrhosis as in controls (P = 0·03–0·007), especially the O‐sensitive, F‐insensitive efflux was increased (+ 225%, P = 0·01–0·006). Fractional F‐sensitive transport was normal in cirrhosis. RBC sodium influx was largely normal in cirrhosis. In conclusion, RBC sodium content is reduced in patients with cirrhosis with a direct relation to serum sodium. Increased RBC sodium efflux is especially related to ouabain‐sensitive, furosemide‐insensitive transport and thus most likely due to upregulated activity of the sodium–potassium pump. The study gives no evidence to an altered intracellular/extracellular sodium ratio or to a reduced fractional furosemide‐sensitive sodium transport in cirrhosis.     

Effects of atriopeptin III on renal function, regional blood flows and left ventricular function in conscious dogs in presence or absence of hypovolaemia

Abstract. The effects of atriopeptin III (AP III) on the left ventricular and renal functions were studied in thirteen chronically instrumented conscious dogs and compared to those of the solvent (saline). In the normovolaemic state, an AP III infusion (1 μg kg^-1 min^-1 i.v.) had no effects on heart rate, on mean arterial or left ventricular pressure, on (dP/dt) Max (2989±119 vs. 3007±155 mmHg s^-1; NS) or on the relaxation rate. The left ventricular endocardial and epicardial coronary blood flows (radioactive microspheres) and the renal flow in the outer cortex (707–683 ml (min^-1 100 g^-1); NS) or in the inner cortex (563–570; NS) were also insignificantly affected by AP III infusion. However, AP III increased urinary flow from 24±6 to 36±7 ml h^-1 (P<0·025) and the Na⁺ and Cl^- excretions by 92 and 98%, respectively, (P<0·025 and P<0·05 vs. saline group) without altering significantly K⁺, urea and creatinine eliminations. In the moderately hypovolaemic state (mean reduction in renal flow: outer cortex -15%; P<0·05, inner cortex -5%; NS), AP III infusion at two doses (1 and 3 μg kg^-1 min^-1) still had no effects on arterial pressure and on the indexes of left ventricular inotropic state and relaxation but in this setting, the diuretic effect of AP III became variable. Five dogs markedly increased their excretion of water, Na⁺ and Cl^- whereas no change was noted in the seven remaining dogs. Regional renal blood flows and urinary output before infusion were similar in the responders and non-responders but the mean arterial pressure (81±2 vs. 73±3 mmHg; P<0·01) was lower in the non-responders. It is concluded that AP III has no effect on left ventricular contractility or on the coronary vasculature; at small doses, its diuretic effects appear independent of a renal vasodilation and are rapidly blunted in the presence of hypotension.     

Mesenteric artery response to head-up tilt-induced centralhypovolaemia and hypotension

Superior mesenteric artery (SMA) blood flow and impedance were evaluated byduplex ultrasound during head-up tilt (HUT)-induced central hypovolaemia and hypotension ineight healthy volunteers. HUT induced a reduction in cardiac stroke volume from88·8±6·3 to 64·7±6·3 ml(mean±SEM; P<0·01) and an increase in thoracic electricimpedance from 38·6±2·1 to 42·6±2·1Ω (P<0·01) reflecting a reduced central blood volume. Maintainedtilt provoked a 30% reduction in mean arterial pressure (from 87·1±3·3to 63·4±3·6 mmHg; P<0·01) and the appearanceof presyncopal symptoms. During both the normotensive and the hypotensive phase of HUT, theSMA diameter (5·7±0·03 mm) and blood flow (514±75 ml min^?1) did not change significantly, although the end-diastolic velocity increasedfrom 9·7±4·8 to 39·7±4·0 cm s^?1 (P<0·01). The increase in diastolic velocity, despite amaintained or reduced arterial pressure, supports a reduction in the SMA impedance as it wasreproduced during a meal test when a moderate reduction in mean arterial pressure (87±4to 80±4 mmHg; P=0·04) was accompanied by a ninefoldincrease in the end-diastolic velocity (P<0·01). The results indicate areduction in the mesenteric vascular impedance to the extent that superior mesenteric artery bloodflow is maintained during HUT-induced central hypovolaemia and hypotension.     

Atrial natriuretic peptide concentrations in umbilical cord plasma from pre-eclamptic women

Summary. Atrial natriuretic peptide (ANP) was measured in arterial and venous umbilical cord plasma at the time of delivery by cesarean section in pre-eclamptic (n= 7) and normal women (n= 6). In addition venous samples were obtained from pre-eclamptic (n= 7) and normal pregnant women (n= 7) near term. ANP plasma levels were higher in pregnant women with pre-eclampsia than in normal pregnant women (27·9±4·4 [mean±SEM] and 14·1 ±2·5 pmol 1^-1, respectively, P<0·05). Immediately after delivery plasma ANP in pre-eclamptic mothers was 66·7 ± 12·8 pmol 1^-1 compared to 13·9 ±2·2 pmol 1^-1 in normal mothers (P<0·01). However, in the pre-eclamptic group the levels of ANP in arterial and venous umbilical cord plasma (19·5 ±4·2 and 16·7±4·3 pmol 1^-1 respectively) were significantly (P<0·01) lower than ANP levels in arterial and venous cord plasma (39·6 ± 1·0 and 31·1±4·2 pmol 1^-1, respectively) from normal mothers. It is concluded that the increased ANP plasma level in pre-eclamptic women originates from a maternal source. In addition, since the ANP level is lower in cord plasma than in maternal plasma in pre-eclampsia, fetoplacental volume homeostasis may also be changed in pre-eclampsia.     

Determination of baroreflex gain using auto-regressive moving-average analysis during spontaneous breathing

The heart rate component of the arterial baroreflex gain (BRG) was determined with auto-regressive moving-average (ARMA) analysis during each of spontaneous (SB) and random breathing (RB) protocols. Ten healthy subjects completed each breathing pattern on two different days in each of two different body positions, supine (SUP) and head-up tilt (HUT). The R–R interval, systolic arterial pressure (SAP) and instantaneous lung volume were recorded continuously. BRG was estimated from the ARMA impulse response relationship of R–R interval to SAP and from the spontaneous sequence method. The results indicated that both the ARMA and spontaneous sequence methods were reproducible (r=0·76 and r=0·85, respectively). As expected, BRG was significantly less in the HUT compared to SUP position for both ARMA (mean ± SEM; 3·5 ± 0·3 versus 11·2 ± 1·4 ms mmHg^–1; P<0·01) and spontaneous sequence analysis (10·3 ± 0·8 versus 31·5 ± 2·3 ms mmHg^–1; P<0·001). However, no significant difference was found between BRG during RB and SB protocols for either ARMA (7·9 ± 1·4 versus 6·7 ± 0·8 ms mmHg^–1; P=0·27) or spontaneous sequence methods (21·8 ± 2·7 versus 20·0 ± 2·1 ms mmHg^–1; P=0·24). BRG was correlated during RB and SB protocols (r=0·80; P<0·0001). ARMA and spontaneous BRG estimates were correlated (r=0·79; P<0·0001), with spontaneous sequence values being consistently larger (P<0·0001). In conclusion, we have shown that ARMA-derived BRG values are reproducible and that they can be determined during SB conditions, making the ARMA method appropriate for use in a wider range of patients.     

Abnormal Na+-K+ ATPase kinetics in a subset of essential hypertensive patients

We have performed a kinetic analysis of the interaction of Na+-K+ ATPase with internal Na+ in erythrocytes of 30 normotensive controls and 72 essential hypertensive patients. Neither the maximal rate of ouabain-sensitive sodium efflux (Vmax) nor the internal Na+ content required for half-maximal stimulation (K50%) were significantly different between normotensive and hypertensive patients. Nevertheless, using the 95% confidence limits of the K50% in the normotensive group as a cut-off point, 13 (18.06%) essential hypertensive patients exhibited increased values of this parameter (29.16 +/- 4.31 mmol l-1 cells) revealing decreased affinity of Na+-K+ ATPase for internal Na+ (Pump-hypertensives). The Vmax was also higher in the Pump '-' subset (14.08 +/- 4.85 mmol (1 cells h)-1 vs. 6.92 +/- 1.80; P = 0.0002) and 10 of these 13 hypertensives exhibited a Vmax above the upper end limit of 10.5 mmol (1 cells h)-1, suggesting a compensatory effect. No differences were observed between the Pump '-' subset and the remaining 59 hypertensives without Na+-K+ pump abnormality when basal erythrocyte Na+ content and clinical parameters of hypertension were examined. Decreased apparent affinity of Na+-K+ pump for internal Na+ present in 9-27% of essential hypertensives may be implicated in pathogenetic mechanisms of hypertension.     

Erythrocyte Na+-H+ exchanger and Na+-Li+ countertransport activity in primary aldosteronism

Abstract. Several authors have described increased Na-H exchanger activity in essential hypertension but no data are available in secondary forms of hypertension such as primary aldosteronism. We measured Na-H exchanger kinetics together with Na-Li countertransport V _max in the erythrocytes of eight patients with primary aldosteronism and in 15 normotensive control subjects. Plasma aldosterone, plasma renin and plasma potassium were also evaluated. Na-H exchanger V _max appear to be increased in patients with primary aldosteronism and Hill's n , an index of co-operativity amongst intracellular proton binding sites, was significantly lower in patients than in controls. No statistically significant differences were found between affinity for intracellular protons (K50%) and for Na-Li countertransport V _max between the two groups studied. We were unable to find any correlations between Na-H exchanger V _max and Na-Li countertransport V _max in the two groups considered as a whole. From the present data Na-H exchanger overactivity would not appear to be a specific feature of essential hypertension but seems to be characteristic in patients with primary aldosteronism.     

Effect of inhibition of the electrogenic Na+/K+ pump on the mechanical activity in the rat uterus

Summary— The effects of ouabain and K⁺-free solution were studied in estrogen-primed rat uterine strips under resting tone or repeatedly stimulated with KCl, acetylcholine or oxytocin applied for 20 minutes at 60 minute intervals. These effects were compared with those of the K⁺ channel opener cromakalim. In preparations under resting tone, ouabain (0.1 mM and 0.3 mM) induced rhythmic contractions which disappeared after 20–30 minutes whereas at a higher concentration (1 mM) it evoked a rapid, phasic response followed by a small tonic contraction. Exposure of the strip to a K⁺-free solution induced either rhythmic waves, which ceased after 8–10 minutes, or a single phasic contraction which was followed by a small and slow increase in the resting tone (54 ± 10 mg after 180 min exposure). Nifedipine (0.3 μM) abolished the rhythmic or phasic component of these responses but failed to modify the late small tonic contraction induced by ouabain 1 mM or by K⁺-free solution. Ouabain (0.1–1 mM) or K⁺-free-evoked responses disappeared after short (4 min) or prolonged (60 min) exposure to a Ca²⁺-free, 3 mM EGTA-containing solution. Cromakalim (10 nM ?0.1 mM) did not induce any variation in the resting tone either in the presence or in the absence of Ca²⁺ in the medium. In strips repeatedly stimulated with acetylcholine (0.1 mM) or oxytocin (1 μM), ouabain (0.3 mM), K⁺-free-solution and cromakalim (10 μM) reduced the amplitude of the initial, phasic response and progressively decreased the oscillatory component of the response to these agonists. Conversely, the successive responses evoked by KCl 60 mM in similar experimental conditions were not affected by ouabain or cromakalim. Ouabain (0.3 mM), K⁺-free solution and cromakalim (10 μM) decreased the Ca²⁺-independent, maintained contractions induced by acetylcholine or oxytocin after prolonged exposure to a Ca²⁺-free, EGTA-containing medium. These inhibitory effects were partially or completely reversed in the presence of the non-selective potassium channel blocker tetraethylammonium (10 mM) or in a Ca²⁺-free solution containing 60 mM K⁺. In conclusion, these results suggest that the response induced by ouabain or K⁺-free solution in estrogen-primed rat myometrium involves Ca²⁺ influx through potential-operated calcium channels but not Ca²⁺ release from intracellular stores. In addition, our results show that prolonged exposure to ouabain or K⁺-free medium decreases membrane receptor-mediated responses in rat uterus. This inhibitory effect seems to be the result, at least in part, of a decrease in the cytosolic level of K⁺, due to the inhibition of the electrogenic Na⁺ pump.     

Cholinergic blockade and the mesenteric artery response to head-up tilt-induced central hypovolaemia

The influence of muscarinic blockade on the superior mesenteric artery (SMA) response to head-up tilt (HUT) was assessed by Doppler ultrasound in eight healthy adults pretreated with i.v. glycopyrron. During supine rest, cholinergic blockade increased heart rate from 58 ± 3 to 106 ± 6 beats min^?1 (mean ± SE) and mean arterial pressure from 81 ± 3 to 97 ± 4 mmHg (P<0·01) and it reduced the cardiac stroke volume from 89 ± 6 to 59 ± 7 ml (P<0·01) with no significant effect on the SMA diameter and blood flow velocities. HUT provoked a further increase in heart rate to 134 ± 5 beats min^?1(P<0·01) and a reduction in stroke volume to 45 ± 4 ml (P<0·01). The early diastolic velocity increased from ?51 ± 4 to 6 ± 8 cm s^?1 during the normotensive stage of HUT and further to 21 ± 9 cm s^?1 during the hypotensive stage with a reduction in mean arterial pressure from 97 ± 4 to 73 ± 7 mmHg (P<0·01) but, in contrast to control HUT (without cholinergic blockade), the end-diastolic velocity did not change significantly. Maintenance of blood velocity and diameter in spite of an increase in arterial pressure at rest indicates increased SMA impedance. Likewise, during hypovolaemia, a glycopyrron-induced inhibition in diastolic velocity supports an increase in SMA impedance. The results indicate cholinergic vasorelaxing influence on the superior mesenteric artery both at rest and during normotensive central hypovolaemia.     

Ovariectomy increases vascular calcification via the OPG/RANKL cytokine signalling pathway

Background Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor‐κΒ ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis. Materials and methods Age‐matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post‐procedure, atherosclerosis was induced by 15 months 0·2%‐cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (µCT) and molecular analysis of the vascular tissue. Results Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8·4 ± 2·8 vs. 1·9 ± 0·6 mg cm^?3, P = 0·042, and 94 ± 26 vs. 33 ± 7 particles cm^?3, P = 0·046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 ± 7° vs. SHAM 33 ± 5°, P = 0·99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0·029, primarily by decreasing RANKL (P = 0·019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study. Conclusions In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ‐specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post‐menopausal women.     

Increased sympathetic tone in forearm subcutaneous tissue in primary hypothyroidism

Summary. Sympathetic reflex regulation of subcutaneous blood flow (SBF) in the forearm was studied in eight patients with primary hypothyroidism. Diastolic arterial pressure was ≥95 mmHg in five patients. SBF was determined by local clearance of Na^99mTcO₄. Sympathetic vasoconstriction normally seen after lowering the forearm 40 cm below heart level was absent since SBF only decreased by 4% (± 7%, P > 0·1) during these conditions. In head-up vertical position we noticed a diminished baroreceptor response as SBF at heart level was reduced by 11% (±7%, P < 0·1) compared to supine position. After proximal local anaesthesia SBF increased by 351% (±81%, P < 0·01) and disclosed a normal vasoconstrictor response as SBF was reduced by 53% (±5%, P < 0·01) during arm lowering. Five of the treated patients were restudied in the euthyroid state. Mean arterial pressure was reduced in mean by 20 mmHg (± 6 mmHg, P < 0·02) during treatment and a significant vasoconstriction was observed both during arm lowering (SBF =-52% (±6%, P<0·02)) and in head-up vertical position (SBF= -45% (± 11%, P<0·02)). In conclusion sympathetic vasoconstrictor activity in adipose tissue is markedly increased in primary hypothyroidism. Sympathetic tone and arterial pressure are reduced during treatment.     

The age‐related reduction in cerebral blood flow affects vertebral artery more than internal carotid artery blood flow

Ageing reduces cerebral blood flow (CBF), while mean arterial pressure (MAP) becomes elevated. According to ‘the selfish brain’ hypothesis of hypertension, a reduction in vertebral artery blood flow (VA) leads to increased sympathetic activity and thus increases MAP. In twenty‐two young (24 ± 3 years; mean ± SD) and eleven elderly (70 ± 5 years) normotensive men, duplex ultrasound evaluated whether the age‐related reduction in CBF affects VA more than internal carotid artery (ICA) blood flow. Pulse‐contour analysis evaluated MAP while near‐infrared spectroscopy determined frontal lobe oxygenation and transcranial Doppler middle cerebral artery mean blood velocity (MCA V_mean). During supine rest, MAP (90 ± 13 versus 78 ± 9 mmHg; P<0·001) was elevated in the older subjects while their frontal lobe oxygenation (68 ± 7% versus 77 ± 7%; P<0·001), MCA V_mean (49 ± 9 versus 60 ± 12 cm s^?1; P = 0·016) and CBF (754 ± 112 versus 900 ± 144 ml min^?1; P = 0·004) were low reflected in VA (138 ± 48 versus 219 ± 50 ml min^?1; P<0·001) rather than in ICA flow (616 ± 96 versus 680 ± 120 ml min^?1; P = 0·099). In conclusion, blood supply to the brain and its oxygenation are affected by ageing and the age‐related decline in VA flow appears to be four times as large as that in ICA and could be important for the age‐related increase in MAP.     

Ascorbic acid reduces the endotoxin-induced lung injury in awake sheep

Abstract Our aim was to investigate whether ascorbic acid can reduce reactive oxygen metabolite-mediated acute lung injury. The effects of intravenous administration of Escherichia coli endotoxin were studied, with and without ascorbic acid infusion, on haemodynamics, lung lymph flow, cardio-respiratory and neutrophil function in chronically instrumented sheep. Paired experiments were performed on eight sheep in which they received either endotoxin alone (0·5 μg kg^-1 b.w.) (ET group) or in combination with an ascorbic acid infusion (1 g kg^-1 b.w. bolus injection followed by 0·2 g kg^-1 h^-1 continuous infusion) (ET + ASC group) in random order. Four of the animals also received ascorbic acid alone (ASC group). As a result, for the ET + ASC group a general and mostly significant improvement (P < 0·05) in the early hypertensive phase (0·60 min, P values) and in the late permeability phase (2–4 h, *P values) of cardiorespiratory function (mean artery pressure: P/*P= 0·283/0·049; mean pulmonary artery pressure: P/*P= 0·0.0001; mean pulmonary artery wedge pressure: P/*P= 0·02/0·001; right ventricular stroke work index: P/*P= 0·02/0·0001; cardiac index: P/*P= 0·797/0·755; arterial oxygen saturation: P/*P= 0·0059/0·01; arterial-venous difference of oxygen tension: P/*P= 0·011/0·0005), oxygen consumption: P/*P=0·013/0·035, lung lymph flow: P/*P=0·562/0·012, lymph/plasma protein ratio: P/*P= 0·304/0·008 and protein clearance: P/*P= 0·56/0·05 was observed in comparison with the ET group. The decrease of the neutrophil chemiluminescence response of the ET + ASC group in the late phase: P/*P= 0·419/0·026 was most likely due to ascorbic acid, whereas β-N-acetylglucosaminidase secretion was identical for both groups. In conclusion, we suggest that reactive oxygen metabolite scavenging by ascorbic acid is responsible for the improvement of endotoxin-induced acute lung injury.     

The effects of beta‐alanine supplementation on physical working capacity at heart rate threshold

Beta‐alanine (BA) supplementation has been shown to delay neuromuscular fatigue as a result of increased muscle carnosine concentrations. Carnosine has also been found in brain and cardiac tissue. The physical working capacity test at heart rate threshold (PWC_HRT) is a global estimate of the onset of fatigue during exercise, influenced by central and peripheral factors. The purpose of this study was to determine the effects of 28 days of BA supplementation on the PWC_HRT. Thirty subjects (mean ± SD; age: 21·0 ± 2·1 years; body mass: 72·7 ± 14·5 kg; height: 170·1 ± 7·9 cm) were randomly assigned to BA (n = 15) or placebo (PL, n = 15) groups. Testing included eight to nine total visits: an enrolment day, physical screening, peak oxygen consumption (V^·O_2peak) and two PWC_HRT assessments over 4 days. Significant differences existed between BA and PL for PWC_HRT (P = 0·001; mean?: BA? = +24·2 watts, PL? = +11·2 watts), but not for V^·O_2peak (P = 0·222), time to exhaustion (TTE; P = 0·562) or ventilatory threshold (VT; P = 0·134). Results suggest that BA may increase heart rate training threshold. These results, in combination with one previous study reporting a potential effect of BA on HR, suggest that future studies should evaluate both central and peripheral aspects of fatigue with BA intake.