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.     

Na+/H+ antiporter activity in peripheral blood lymphocytes of obese and type 2 diabetic patients is increased only in the presence of arterial hypertension

Abstract. The aim of this work is to evaluate whether type 2 diabetes mellitus, obesity and arterial hypertension, three conditions characterized by the presence of insulin resistance, share some common genetic markers. A potential candidate is the Na⁺/H⁺ anti-porter, the increased activity of which is considered a marker of essential hypertension. This ion exchanger seems to be related to the Na⁺/Li⁺ countertransport, that is considered a marker of insulin resistance in essential hypertension and in type 1 diabetes mellitus. In this study we wished to clarify whether the activity of the Na⁺/H⁺ antiporter is increased not only in hypertensive subjects, but also in obese and type 2 diabetic patients, both in the presence and in the absence of arterial hypertension. The activity of the ion exchanger was measured in peripheral blood lymphocytes (PBL) by clamping intracellular pH (pH_i) at 5·8–6·2 and then detecting the rate of the proton efflux after sodium addition. In the absence of arterial hypertension, no significant difference in this parameter was observed in obese and type 2 diabetic patients in comparison with normal subjects. In the presence of arterial hypertension, there was a significant increase in the Na⁺-induced H⁺ efflux at the internal pH (pHi) values of 5·8 and 6·2 both in hypertensive controls and in hypertensive obese and type 2 diabetic patients (P= 0·05–0·0001 vs. normotensive subjects and patients). In particular, H⁺ efflux at pH 5·8 (mmol l^-1 min^-1) was 35·36 ± 2·48 in normotensive and 42·77 ± 1·63 in hypertensive control subjects (P= 0·045), 33·06 ± 1·88 in normotensive and 50·40 ± 5·21 in hypertensive obese patients (P= 0·009), 31·16 ± 1·84 in normotensive and 55·54 ± 5·83 in hypertensive type 2 diabetic patients (P= 0·0001). H⁺ efflux showed a significant correlation with both systolic (at pHi 5·8, r = 0·473, P= 0·001; at pHi 6·2, r = 0·357, P= 0·016) and diastolic blood pressure (at pHi 5·8, r = 0·600, P= 0·0001; at pHi 6·2, r = 0·555, P= 0·0001). Therefore, our study demonstrates that the hyperactivity of the Na⁺/H⁺ exchanger in peripheral blood lymphocytes is also a marker of arterial hypertension in obesity and in type 2 diabetes mellitus, and that the exchanger activity is not increased in these two conditions in the absence of arterial hypertension.     

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.     

Intracellular pH,intrauterine growth and the insulin resistance syndrome

Defects of both sodium–hydrogen exchange (NHE) and sodium–lithium countertransport (SLC) have been described in subjects at increased risk of coronary heart disease (CHD). Sodium transport is linked to the regulation of cell volume, intracellular pH and cell growth, which may explain aspects of this association. However, impaired growth in early life is also linked to adult CHD, and ‘programmed’ alterations of cell behaviour are postulated to be responsible for this. In this study, therefore, we examined whether NHE or SLC in adults are predicted by anthropometric measures at birth, as well as being associated with insulin resistance syndrome (IRS) variables in adulthood. Red cell SLC was measured in 26 adults, and NHE in dermal fibroblasts from another 15 subjects characterized anthropometrically at birth. SLC activity correlated with LDL cholesterol, triglycerides and urate (r=0·42 – 0·49; 0·05 > P>0·01), but not birth anthropometry. NHE V_max correlated with plasma insulin (r=0·80; P<0·001), but birth weight was unrelated to V_max, K_m or Hill coefficient for H⁺_i. However, pH_i correlated with birth weight (r=0·74; P=0·002), insulin sensitivity (r=0·52; P<0·05), fasting glucose (r=–0·52; P<0·05) 2 h insulin (r=0·51; P<0·05) 2 h glucose (r=–0·54; P<0·05). In conclusion, red cell SLC is related to IRS variables, but not with birth weight measures. In contrast, low intracellular pH_i is related to both low birth weight and adult insulin resistance, suggesting it might be a ‘programmed’ cell phenotype, although this is not apparently explained by altered NHE kinetics.     

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.     

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.     

PAR-1-dependent pp60src activation is dependent on protein kinase C and increased [Ca2+]i: evidence that pp60src does not regulate PAR-1-dependent Ca2+ entry in human platelets

Summary. Background: The role of the tyrosine kinase pp60^src in PAR‐1‐dependent Ca²⁺ entry was investigated in human platelets. pp60^src plays a role in thapsigargin (TG)‐evoked store‐operated Ca²⁺ entry (SOCE), which is thought to be a major component of thrombin‐evoked Ca²⁺ entry. Methods: pp60^src tyr⁴¹⁶ phosphorylation was used to assess pp60^src activation. Fura‐2‐loaded platelets were used to monitor intracellular Ca²⁺ concentration ([Ca²⁺]_i). Results: Activation of PAR‐1 with the specific agonist SFLLRN increased pp60^src activation within 10 s. This required phospholipase C (PLC) activity, Ca²⁺ release and a rise in intracellular Ca²⁺. PP2, an inhibitor of Src‐family tyrosine kinases, inhibited SFLLRN‐evoked Ca²⁺ entry, but also inhibited Ca²⁺ release and the extrusion of Ca²⁺ by the plasma membrane Ca²⁺ ATPase. Actin polymerization and conventional protein kinase C (cPKC) activity were required for TG‐ and SFLLRN‐evoked pp60^src activation. Although Gö6976, an inhibitor of cPKCs, inhibited TG‐evoked SOCE, it had little effect on SFLLRN‐ or thrombin‐evoked Ca²⁺ entry. Conclusions: These data indicate that stimulation of PAR‐1 leads to activation of pp60^src in human platelets, through PLC and cPKC activation, Ca²⁺ release and actin polymerization. However, as PKC and actin polymerization are not needed for SFLLRN‐evoked Ca²⁺ entry, we suggest that pp60^src is also not required. The apparent inhibition of SFLLRN‐evoked Ca²⁺ entry by PP2 is likely to be secondary to reduced Ca²⁺ release. These data argue against a contribution of this SOCE pathway to PAR‐1‐dependent Ca²⁺ entry.     

Hyperchloremic acidosis during grand mal seizure lactic acidosis

Objective To evaluate the prevalence and the mechanism of hyperchloremic acidosis component (HC1A) during lactic acidosis secondary to grand mal seizures. Design Retrospective study. Setting Medical intensive care unit in a university hospital. Patients 35 patients admitted for grand mal seizures with lactic acidosis (pH<7.35, TCO₂ <20 mmol/l and PaCO₂ <8 kPa). Measurements HC1A was defined by the ratio: excess anion gap/HCO₃ deficit (ΔAG/ΔTCO₂) <0.8. A difference in the distribution space of protons and their accompanying anion, i.e., a displacement of chloride from cells by the entering lactate, was evaluated by the ratio natremia/chloremia (Na⁺/Cl⁻). Results Immediately after seizures, a profound lactic acidosis was observed (pH=7.22±0.17 (mean±SD), AG: 23.8±7.1 mmol/l, TCO₂=14.5±5.3 mmol/l, lactate: 14.6±6.9 mmol/. HC1A was present on admission in 11 patients (31.5%). Its prevalance increased to 73% after recovery. ΔAG/ΔTCO₂ ratios were unrelated to creatinine, level and PaCO₂, but dependent on the ratio Na⁺/Cl⁻ (r=0.803;p<0.001, ΔAG/ΔTCO₂=6.4 X (Na⁺/Cl⁻)−7.9). These data demonstrate that HC1A is not a respiratory or renal phenomenon and suggest differences in the distribution spaces of hydrogen ions and their accompanying anions. Conclusion HC1A component may be associated with lactic acidosis in grand mal seizures and appears to be secondary to a lactate antiport. This phenomenon could be an immediate physiological response to a sudden metabolic acidosis.     

Interactions of stress hormones on lipid and carbohydrate metabolism in man with partial insulin deficiency

Abstract. The metabolic responses to 4-h infusions of adrenaline (3 μg kg^-1 h^-1) and cortisol (10 mg m^-2 h^-1 for 2 h followed by 5 mg m^-2 h^-1 for 2 h), separately and in combination, have been studied in six healthy subjects with concurrent somatostatin infusion (250 μg h^-1). A combined infusion of adrenaline, cortisol, glucagon (180 ng kg^-1 h^-1) and somatostatin has also been studied. Somatostatin plus adrenaline and somatostatin plus cortisol resulted in hyperglycaemia (at 240 min, somatostatin plus adrenaline 11·4 ± 0·4 mmol l^-1, P < 0·001; somatostatin plus cortisol 6·7 ± 0·3 mmol l^-1, P < 0·05; somatostatin alone 4·9 ± 0·4 mmol l^-1). No synergistic effect on blood glucose was seen with adrenaline and cortisol together. When glucagon was added, blood glucose rose more rapidly than without glucagon (9·3 ± 0·4 mmol l^-1v. 7·2 ± 0·5 mmol l^-1 at 45 min, P < 0·001), but plateau values were similar. Plasma NEFA levels were raised by somatostatin plus adrenaline (0·55 ± 0·04–1·82 ± 0·11 mmol l^-1 at 60 min). Somatostatin plus cortisol had no more effect on plasma NEFA than somatostatin alone. During the combined infusion of somatostatin plus adrenaline plus cortisol, a synergistic effect on plasma NEFA was observed (2·30 ± 0·11 mmol l^-1 at 60 min, P < 0·01 v. somatostatin plus adrenaline). This occurred despite a small escape of insulin secretion. The lipolytic actions of adrenaline are potentiated by elevated circulating cortisol levels in insulin-deficient man. Glucagon does not modify this response, but accelerates the development of hyperglycaemia.     

Interstitial colloid osmotic and hydrostatic pressures in human subcutaneous tissue during early stages of heart failure

Summary. Subcutaneous oedema is a common finding in heart failure. However, some patients have reduced cardiac pump function without oedema. The aim of this study was to investigate whether local mechanisms in subcutaneous tissue contribute to oedema prevention. A reduction in interstitial colloid osmotic pressure (φ_i) and a rise in interstitial fluid hydrostatic pressure (P_i) will both counteract a rise in capillary filtration caused by heart failure. Cardiac catheterization was done in 22 angina pectoris patients without visible oedema. Two days later φ_i was measured with a wick method and P_i was measured with a wick-in-needle method. Both parameters were measured in subcutaneous tissue on thorax at heart level and on the ankle. Plasma volume was determined by ¹²⁵I-albumin and extracellular volume measured with ³⁵SO₄. Parameters of cardiac pump function ranged from normal to clearly pathological values. Mean φ_i was 13·0 mmHg on thorax and 8·3 mmHg on the ankle. P_i, averaged -2·1 mmHg on thorax and -1·5 mmHg on the ankle. Statistically significant (P<0·05) correlations were found between φ_i on thorax and left ventricular end diastolic pressure (r_s -0·40) and φ_i on thorax and cardiac index (r_s 0·42). P_i, was positively correlated to right atrial pressure (r_s 0·50). Body fluid volumes were normal or moderately reduced. The study shows that a reduction in cardiac pump function is associated with a reduction in φ_i and a rise in P_i. These changes may help to prevent oedema formation in the early stages of heart failure.     

A Drosophila melanogaster hobo-white+ vector mediates low frequency gene transfer in D. virilis with full interspecific white+ complementation

Transformation of a Drosophila virilis white mutant host strain was attempted using a hobo vector containing the D. melanogaster mini-white⁺ cassette (H[w⁺, hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (HFL1), one containing only the white⁺ marked vector, and a sibling line containing the vector as well as an HFL1 helper integration. An approximate total transformation frequency of 1% is deduced. A high frequency of wing and eye morphology mutants were also observed, suggesting that hobo may have mobilized a related element in D. virilis. The data reaffirms a relatively low transformation vector activity for the hobo transposon in D. virilis; however, nearly full interspecific expression of the white⁺ marker supports its possible function in other species as well.     

Inter-relations between the calcium set-points of Parfitt and Brown in primary hyperparathyroidism: a sequential citrate and calcium clamp study

Abstract. The objective of the present study was to compare the calcium set-points of E. M. Brown and A. M. Parfitt obtained by sequential citrate and calcium clamp in patients with primary hyperparathyroidism and healthy controls. Twenty-six patients with primary hyperparathyroidism were investigated and compared to 22 healthy volunteers. All participants were investigated by sequential calcium lowering and raising comprising the following four phases: Phase (1) blood ionized calcium lowering of about 0·20 mmol l^-1; phase (2) steady-state (relative) hypocalcaemia of blood ionized calcium 0·20 mmol l^-1 below baseline; phase (3) blood ionized calcium is raised to about 0·20 mmol l^-1 above baseline; and phase (4) (relative) hypercalcaemia of blood ionized calcium 0·20 mmol l^-1 above baseline. Serum parathyroid hormone (1–84) was measured by an immunoradiometric assay. Blood ionized calcium was measured by a calcium selective electrode. We found the calcium set-points of Parfitt to be 1·42 mmol l^-1 (SD 0·12, n= 52) vs. 1·25 mmol l^-1 (SD 0·04, n= 44) in patients and controls, respectively (P < 0·001). The calcium set-points of Brown were 1·32 mmol l^-1 (SD 0·10, n= 26) vs. 1·13 mmol l^-1 (SD 0·04, n= 22), respectively (P < 0·001). By comparing the calcium set-points of Parfitt and Brown, a strikingly good correlation was observed, in patients (r= 0·91, P < 0·001) and in controls (r= 0·85, P < 0·001). We demonstrate in this paper in vivo that Brown's and Parfitt's calcium set-points are raised in primary hyperparathyroidism and return to normal following parathyroidectomy. The values for Brown's and Parfitt's calcium set-points are significantly different, but strikingly well correlated, supporting the view that Brown and Parfitt describe two different points on the same sigmoidal curve, corresponding to 50% and about 85% inhibition of PTH maximum, respectively. The mathematical form of the sigmoidal curve between blood ionized calcium and parathyroid hormone is very similar in primary hyperparathyroidism and normal humans.     

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.     

Plasma lipid and lipoprotein profile in elderly female runners

Plasma lipid and lipoprotein profiles were compared in elderly female runners (RU: n= 15, aged 66 ± 5 years, body fat 20 ± 4%, training distance 35 ± 15 km week¹, V?O_2max 36 ± 4 ml kg¹ min¹, mean ± SD) and age-matched untrained women (UT: n= 28, 66 ± 4 years, body fat 26 ± 6%, V?O_2max 26 ± 3 ml kg¹ min¹). There were insignificant differences in total cholesterol (RU: 5·04 ± 0·60 vs. UT: 5·48 ± 0·85 mmol 1¹), HDL-cholesterol (RU: 1·97 ± 0·41 vs. UT: 1·91 ± 0·36 mmol 1¹) and LDL-cholesterol (RU: 2·72 ± 0·59 vs. UT: 3·03 ± 0·80 mmol 1¹) between the two groups. Plasma triglyceride concentration of the runners was significantly lower than that of the untrained women (RU: 0·80 ± 0·27 vs UT: 1·14 ± 0·36 mmol 1¹, P < 0·01). No difference was observed in the LDL-cholesterol/HDL-cholesterol ratio between the two groups (RU: 1·45 ± 0·51 vs UT: 1·64 ± 0·53 units). These results suggest that regularly performed running of 35 km week¹ in elderly women does not further elevate their HDL-cholesterol level which is already high compared to the levels found in elderly men. However, elderly female runners appear to be protected against age-related increases in the levels of triglyceride and LDL-cholesterol.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

Influence of the endothelium on Ca2+ dependency of angiotensin II-induced contractions of rat aortic rings

Summary— Endothelium-dependent relaxation has been demonstrated to be involved in the regulation of vascular tone and extracellular Ca²⁺ was found to play a prominent role in this process. Since the dependency on extracellular Ca²⁺ appeared to differ considerably within the arterial tree, possibly as a consequence of vessel-related endothelium-dependent mechanisms, we investigated the effects of different compounds affecting Ca²⁺ entry (nifedipine, CoCl₂) on angiotensin II-induced contractions of rat aortic rings with and without endothelium as well as the responses in a Ca²⁺–“free” solution. For this purpose, rat aortic rings were either undone from their endothelial layer by gentle mechanical rubbing or care was taken to keep the intima intact in case rings where endothelium were required. The presence of an intact endothelium was confirmed by acetylcholine-induced relaxation. A stronger responsiveness towards angiotensin I, both after a complete concentration-response curve and after a single maximal concentration of angiotensin II was observed in arterial segments without endothelium. The maximal contraction to a single concentration of angiotensin II (0.1 μM) in the rings without endothelium amounted to 75.8 ± 3.8% of the preceding response to a supramaximal concentration of noradrenaline (= E_max). In rings without the endothelial layer, the contraction was 34.8 ± 3.7% of E_max. This indicates an endothelium-induced relaxation in aortic rings with endothelium. After incubation with the Ca²⁺ entry blocker nifedipine (1 μM) both rings with and without endothelium were inhibited to the same extent, contractions amounted to 30.7 ± 1.8% and 19.6 ± 1.3% of E_max, respectively. However, incubation in a Ca²⁺-“free” medium for 5 min resulted in similar contractions for rings without endothelium (16.4 ± 1.4% of E_max) as for rings with endothelium (15.0 ± 1.6% of E_max). Moreover, CoCl₂ in a concentration of 300 μM hardly inhibited the contraction of rings with an intact endothelium, a contractile response of 30.5 ± 2.8% of E_max was observed. The results of the study suggest that the influx of Ca²⁺ions is indeed responsible for the endothelium-mediated relaxation. However, this influx, which cannot be antagonized by nifedipine, but has shown to be affected by CoCl₂, suggests that channels intensitive to organic Ca²⁺ entry blockers may be involved.     

Water diuretic effect of intravenously administered 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in conscious man

Summary. The stable prostaglandin analogue 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ (9-methylene-PGE₂) was infused intravenously (0·5 ml/min) in the dosage of 20 μg/min for 2 h in conscious euhydrated man. The administration of 9-methylene-PGE₂ rapidly induced an increase in urine flow (from 1·2±0·07 to 5·35±1·07 ml/min) concomitantly with a decrease in urine osmolality (from 827±40 to 193±44 mOsm/kg). Parallel to this tubular reabsorption of sodium (Na^f), calcium (Ca²⁺) and magnesium (Mg³⁺) increased and that of potassium (K⁺) decreased as shown by a reduction in the clearance for respective ion divided by the clearance of inulin. Apparently the water diuresis was mediated by an inhibition of arginine vasopressin's (AVP) antidiuretic effect. The mechanism behind the increase in renal tubular reabsorbtion of Na⁺ could possibly be a 9-methylene-PGE₂ mediated modulation of the renal aldosterone effect. However the protocol followed did not provide any evidence for this, or any other explanation of the observed renal retention of Na⁺, Ca²⁺ and Mg²⁺. The results reported here indicate that 9-methylene-PGE₂ may have a future use as a water diuretic agent in patients suffering from water retention and dilutional hyponatraemia such as seen in the syndrome of inappropriate antidiuretic hormone (AVP) release commonly known as SIADH or Schwartz-Bartter's Syndrome.     

The increase in CO2 production induced by NaHCO3 depends on blood albumin and hemoglobin concentrations

Objective: To evaluate the origin of H⁺ ions participating in the generation of CO₂ coming from sodium bicarbonate infusion during metabolic acidosis. We hypothesized that these H⁺ ions come from a back-titration of the main non-bicarbonate buffers present in the blood, i. e. the hemoglobin and the albumin, and thus postulated that the rate of CO₂ release from a bicarbonate load is dependent on the concentration of these buffers. Design: Prospective clinical and experimental study. Setting: Surgical intensive care unit of a university hospital Patientsand material: (1) Sixteen stable sedated and artificially ventilated critically ill patients with a mild base deficit. (2) Acidotic human blood (bicarbonate 5 mM, pH 7.0) of hematocrit 5, 10, 20 and 40 % regenerated from a mixture of frozen fresh plasma and packed red blood cells. Interventions: Patients: infusion of 1.5 mmol/kg sodium bicarbonate over 5 min. Regenerated blood: 25 mM sodium bicarbonate load. Measurements and results: Patients: continuous measurement of CO₂ production (VCO₂) on the expired gas using a metabolic monitor and arterial blood gas analysis before (T0), at the end (T5) and at 10, 30 and 60 min after the beginning of the bicarbonate infusion. The increase in VCO₂ was 18 ± 7 % leading to a rise in PaCO₂ from 39.6 ± 2.3 at T0 to 46.2 ± 2.7 mmHg at T5. The increases in VCO₂ and in PaCO₂ were significantly correlated to the albumin (r = 0.73, p < 0.005 and r = 0.70, p < 0.005, respectively) and to the hemoglobin (r = 0.51, p < 0.05 and r = 0.65, p < 0.01, respectively) concentrations. Regenerated blood: gas analysis 1 min after the bicarbonate load. The increase in PCO₂ was closely related to the hematocrit (Ht) of the blood as it was 15.9 ± 7.5 mmHg for Ht 5 %, 29.0 ± 9.6 for Ht 10 %, 44.2 ± 5.9 for Ht 20 % and 71.0 ± 3.5 for Ht 40 % (n = 5 for each, p < 0.001). Conclusions: The importance of the release of CO₂ from a bicarbonate load is dependent on the concentration of the blood non-bicarbonate buffers. It is therefore likely that the adverse effects of bicarbonate therapy linked to the CO₂ generation are more important in patients with high blood albumin and hemoglobin concentrations. Received: 2 August 1999 Final revision received: 1 February 2000 Accepted: 25 February 2000