Hypoxic preconditioning attenuates lipopolysaccharide-induced oxidative stress in rat kidneys

Chronic hypoxic (CH) preconditioning reduces superoxide-induced renal dysfunction via the upregulation of superoxide dismutase (SOD) activity and contents. Endotoxaemia reduces renal antioxidant status. We hypothesize that CH preconditioning might protect the kidney from subsequent endotoxaemia-induced oxidative injury. Endotoxaemia was induced by intraperitoneal injection of lipopolysaccharide (LPS; 4 mg kg⁻¹) in rats kept at sea level (SL) and rats with CH in an altitude chamber (5500 m for 15 h day⁻¹) for 4 weeks. LPS enhanced xanthine oxidase (XO) and gp91phox (catalytic subunit of NADPH oxidase) expression associated with burst amount of superoxide production from the SL kidney surface and renal venous blood detected by lucigenin-enhanced chemiluminescence. LPS induced a morphologic-independent renal dysfunction in baseline and acute saline loading stages and increased renal IL-1β protein and urinary protein concentration in the SL rats. After 4 weeks of induction, CH significantly increased Cu/ZnSOD, MnSOD and catalase expression (16 ± 17, 128 ± 35 and 48 ± 21, respectively) in renal cortex, and depressed renal cortex XO (44 ± 16%) and renal cortex (20 ± 9%) and medulla (28 ± 11%) gp91phox when compared with SL rats. The combined effect of enhanced antioxidant proteins and depressed oxidative proteins significantly reduced LPS-enhanced superoxide production, renal XO and gp91phox expression, renal IL-1β production, and urinary protein level. CH also ameliorated LPS-induced renal dysfunction in the baseline and acute saline loading periods. We conclude that CH treatment enhances the intrarenal antioxidant/oxidative protein ratio to overcome endotoxaemia-induced reactive oxygen species formation and inflammatory cytokine release.     

Differential effect of angiotensin II on blood circulation in the renal medulla and cortex of anaesthetised rats

The renal medulla is sensitive to hypoxia, and a depression of medullary circulation, e.g. in response to angiotensin II (Ang II), could endanger the function of this zone. Earlier data on Ang II effects on medullary vasculature were contradictory. The effects of Ang II on total renal blood flow (RBF), and cortical and medullary blood flow (CBF and MBF: by laser-Doppler flux) were studied in anaesthetised rats. Ang II infusion (30 ng kg⁻¹ min⁻¹ i.v. ) decreased RBF 27 ± 2 % (mean ± s.e.m. ), whereas MBF increased 12 ± 2 % (both P < 0.001). Non-selective blockade of Ang II receptors with saralasin (3 μg kg⁻¹ min⁻¹ i.v. ) increased RBF 12 ± 2 % and decreased MBF 8 ± 2 % ( P < 0.001). Blockade of AT₁ receptors with losartan (10 mg kg⁻¹) increased CBF 10 ± 2 % ( P < 0.002) and did not change MBF. Losartan given during Ang II infusion significantly increased RBF (53 ± 7 %) and decreased MBF (27 ± 7 %). Blockade of AT₂ receptors with PD 123319 (50 μg kg⁻¹ min⁻¹ i.v. ) did not change CBF or MBF. Intramedullary infusion of PD 123319 (10 μg min⁻¹) superimposed on intravenous Ang II infusion did not change RBF, but slightly decreased MBF (4 ± 2 %, P < 0.05). We conclude that in anaesthetised surgically prepared rats, exogenous or endogenous Ang II may not depress medullary circulation. In contrast to the usual vasoconstriction in the cortex, vasodilatation was observed, possibly related to secondary activation of vasodilator paracrine agents rather than to a direct action via AT₂ receptors.     

Decreased peripheral blood γδ T cells in patients with bronchial asthma

Many cell populations are thought to be involved in the etiopathogenesis of bronchial asthma. We examined by flow cytometry the relative and absolute number of CD3*, CD4*, CD8*γδ TcR* T cells. CD19* B cells; and CD56* natural killer (NK) cells in the peripheral blood of 26 adult patients with difficult-to-control asthma (DCA) and 22 patients with minimally symptomatic asthma (MSA). Statistically higher relative and absolute numbers of NK cells (18.39±10.67% and 0.38±0.17×10⁹/l) in comparison with healthy controls (ll.77±8.06% and 0.25±0.19×10⁹/l) and significantly decreased relative and absolute numbers of γδ T cells (3.02±2.16% and 0.06±0.04×10⁹/l) in comparison with controls (5.65+2.90% and 0.13±0.08×10⁹/l) in the DCA patient group were found. After pooling of data from both MSA and DCA patients and dividing the patients according to the presence of allergy, the relative and absolute numbers of 78 T celts were found to be diminished in both the allergy (3.77±2.98 and O.O7±0.O5 ×10⁹/l) and nonallergy (3.06±1.78% and 0.06±0.03 ×10⁹/l) groups in comparison with healthy controls. The reason for the low number of 78 T cells in the peripheral blood of patients suffering from bronchial asthma is under investigation.     

Increased Number of IgG Fc Receptors on Monocyte-Enriched Peripheral Blood Leucocytes from Patients with Rheumatoid Arthritis

The number of free Fc receptors (FcR) per cell and the association constant (K_ass) for the binding of monomeric IgG were determined for monocyte-enriched peripheral blood mononuclear cells, isolated from 16 patients with active classical rheumatoid arthritis (RA) and from 15 normal healthy donors. The assay system was based on binding under equili brium conditions of ¹²⁵I-labelled monomeric rabbit IgG to monocytes purified from peripheral blood on a continuous gradient of Petcoll. Monocytes from 14 untreated RA patients (6 seropositive, 8 seronegative) expressed on the average 4.8±1.3 × 10⁴ FcR/cell. This number was significantly higher (P<0.01) than that found in the control group (3.46±0.7 × 10⁴ FcR/cell). There was also a significant difference between the mean K _ass of the RA group and the control group-2.1±0.7 × 10³1/mol and 2.6±1.0 × 10³ 1/mol, respectively (0.05 >P> 0.01). Two seropositive RA patients receiving systemic treatment with penicillamine expressed the same number of FcR/cell as the mean of the control group (3.6 ± 10⁴). Levels of circulating immune complexes (CIC) and of the complement-factor C3 split product C3d were also measured. No correlation was found between the number of FcR/cell and the concentration of C3d, but there was a weak correlation between the number of FcR/ccll and the level of CIC.     

Renal (pro)renin receptor upregulation in diabetic rats through enhanced angiotensin AT1 receptor and NADPH oxidase activity

Recent studies have demonstrated the presence of the (pro)renin receptor (PRR) in the glomerular mesangium and the subendothelial layer of the renal arteries. We hypothesized that diabetes upregulates PRR expression through enhanced angiotensin subtype 1 (AT₁) receptor–NADPH oxidase cascade activity. Using real-time polymerase chain reaction, Western blot analysis and immunostaining, we studied renal localization of the PRR in the streptozotocin-induced diabetic rat model and in response to 1 week of treatment with the AT₁ receptor blocker valsartan (10 mg kg⁻¹ day⁻¹), the angiotensin AT₂ receptor blocker PD123319 (0.5 mg kg⁻¹ day⁻¹) or the NADPH oxidase inhibitor diphenylene iodonium (DPI; 0.5 mg kg⁻¹ day⁻¹) 6 weeks post-induction of diabetes. Both PRR mRNA and protein were expressed constitutively in the kidneys of normal rat renal cortex and medulla, mainly in glomerular mesangium, proximal, distal and collecting tubules. Compared with normal rats (100%), diabetic rats demonstrated an increase in renal PRR mRNA (184%), protein (228%) and immunostaining. Valsartan and DPI prevented the increase in the PRR mRNA (106 and 126%, respectively), protein (97 and 140%, respectively) and immunostaining that was seen in the kidneys of diabetic rats. The AT₂ blocker PD123319 did not have significant effects on PRR mRNA (157%) or protein expression (200%) in the kidneys of diabetic rats. These results demonstrate that the PRR is constitutively expressed in renal glomeruli and tubules. Expression of the PRR is upregulated in diabetes via enhancement of AT₁ receptor–NADPH oxidase activity.     

Phrenic long-term facilitation requires NMDA receptors in the phrenic motonucleus in rats

Exposure to episodic hypoxia induces a persistent augmentation of respiratory activity, known as long-term facilitation (LTF). LTF of phrenic nerve activity has been reported to require serotonin receptor activation and protein syntheses. However, the underlying cellular mechanism still remains poorly understood. NMDA receptors play key roles in synaptic plasticity (e.g. some forms of hippocampal long-term potentiation). The present study was designed to examine the role of NMDA receptors in phrenic LTF and test if the relevant receptors are located in the phrenic motonucleus. Integrated phrenic nerve activity was measured in anaesthetized, vagotomized, neuromuscularly blocked and artificially ventilated rats before, during and after three episodes of 5 min isocapnic hypoxia ( P _a,O2= 30–45 mmHg), separated by 5 min hyperoxia (50% O₂). Either saline (as control) or the NMDA receptor antagonist MK-801 (0.2 mg kg⁻¹, i.p. ) was systemically injected ∼1 h before hypoxia. Phrenic LTF was eliminated by the MK-801 injection (vehicle, 32.8 ± 3.7% above baseline in phrenic amplitude at 60 min post-hypoxia; MK-801, −0.5 ± 4.1%, means ± s.e.m. ), with little change in both the CO₂-apnoeic threshold and the hypoxic phrenic response (HPR). Vehicle (saline, 5 × 100 nl) or MK-801 (10 μ m ; 5 × 100 nl) was also microinjected into the phrenic motonucleus region in other groups. Phrenic LTF was eliminated by the MK-801 microinjection (vehicle, 34.2 ± 3.4%; MK-801, −2.5 ± 2.8%), with minimal change in HPR. Collectively, these results suggest that the activation of NMDA receptors in the phrenic motonucleus is required for the episodic hypoxia-induced phrenic LTF.     

Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero

In the rat, protein restriction during pregnancy increases offspring blood pressure by 20–30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo . Hence, we hypothesized that exposure to a maternal low-protein diet increases glomerular Ang II AT₁ receptor expression and decreases AT₂ receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT₁ and AT₂ receptor expression, ¹²⁵I-Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT₁ receptor expression was increased (62%) and AT₂ expression was decreased (35%) in LP rats. Maximum ¹²⁵I-Ang II (¹²⁵I-Ang II) binding ( B _max) was increased in LP rats (control n = 9, 291.6 ± 27.4 versus LP n = 7, 445.7 ± 27.4 fmol (mg glomerular protein)⁻¹, P < 0.01), but affinity ( K _D) was not statistically different from controls (control 2.87 ± 0.85 versus LP 0.84 ± 0.20 pmol ¹²⁵I-Ang II, P = 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT₁ receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo . This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.     

Furosemide-induced renal medullary hypoperfusion in the rat: role of tissue tonicity, prostaglandins and angiotensin II

Furosemide (frusemide)-induced renal medullary hypoperfusion provides a model for studies of the dependence of local circulation on tissue tonicity. We examined the role of medullary prostaglandins (PG) and adenosine (Ado) as possible mediators of the response to furosemide. Furosemide was infused i.v. at 0.25 mg kg⁻¹ h⁻¹ in anaesthetized rats, untreated or treated with intramedullary indomethacin (Indo) or Ado. An integrated set-up was used to measure renal medullary laser-Doppler flux (MBF) and medullary ionic tonicity (electrical admittance, Y), and to infuse Indo and Ado directly into the medulla. The cortical flux was measured on kidney surface. The excretion of water, sodium and total solute was also determined. Intramedullary Indo (1 mg kg⁻¹ h⁻¹) decreased MBF 18 ± 5% and increased tissue Y 14 ± 3% (both significant); the treatment abolished the post-furosemide decrease in MBF (−22% in untreated group) and enhanced slightly the increase in renal excretion. Intramedullary Ado (5 mg kg⁻¹ h⁻¹) did not change baseline MBF or Y; the post-furosemide decreases in MBF (−22%) and Y, and the increase in renal excretion were preserved. We conclude that a decrease in intramedullary PG activity secondary to decreased medullary hypertonicity mediates the fall in medullary perfusion in response to furosemide; the hypoperfusion may help restore the initial tonicity. Together with the earlier evidence on the dependence of post-furosemide medullary hypoperfusion on angiotensin II, the study exposes its interaction with PG in the control of medullary circulation. Adenosine is not involved in medullary vascular responses to decreased tissue hypertonicity.     

Maternal nutrient restriction in sheep: hypertension and decreased nephron number in offspring at 9 months of age

Pregnant ewes were fed either a 50% nutrient-restricted (NR; n = 8) or a control 100% (C; n = 8) diet from day 28 to day 78 of gestation (dGA; term = 150 dGA). Lambs were born naturally, and fed to appetite throughout the study period. At 245 ± 1 days postnatal age (DPNA), offspring were instrumented for blood pressure measurements, with tissue collection at 270 DPNA. Protein expression was assessed using Western blot, glomerulus number determined via acid maceration and hormone changes by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). NR lambs had higher mean arterial pressure (MAP; 89.0 ± 6.6 versus 73.4 ± 1.6 mmHg; P < 0.05), fewer renal glomeruli (57.8 ± 23.8 versus 64.6 ± 19.3 × 10⁴; P < 0.05), increased expression of angiotensin converting enzyme (ACE) in the renal cortex (942 ± 130 versus 464 ± 60 arbitrary pixel units (apu); P < 0.03), and increased angiotensin II receptor AT2 expression in the renal medulla (63.3 ± 12.1 versus 19.5 ± 44.2 × 10⁴ apu; P < 0.03). All data are presented as mean ± s.e.m. The present data indicate that global maternal nutrient restriction (50%) during early to mid-gestation impairs renal nephrogenesis, increases MAP, and alters expression of AT2 and ACE without an associated change in birth weight. These data demonstrate the existence of a critical window of fetal susceptibility during early to mid-gestation that alters kidney development and blood pressure regulation in later life.     

Fuel selection in Wistar rats exposed to cold: shivering thermogenesis diverts fatty acids from re-esterification to oxidation

This study characterizes the effects of shivering thermogenesis on metabolic fuel selection in Wistar rats. Because lipids account for most of the heat produced, we have investigated: (1) whether the rate of appearance of non-esterified fatty acids ( R _a NEFAs) is stimulated by shivering, (2) whether mono-unsaturated (oleate) and saturated fatty acids (palmitate) are affected similarly, and (3) whether the partitioning between fatty acid oxidation and re-esterification is altered by cold exposure. Fuel oxidation was measured by indirect calorimetry and fatty acid mobilization by continuous infusion of 9,10-[³H]oleate and 1-[¹⁴C]palmitate. During steady-state cold exposure, results show that total heat production is unequally shared by the oxidation of lipids (52% of metabolic rate), carbohydrates (35%) and proteins (13%), and that the same fuel selection pattern is observed at all shivering intensities. All previous research shows that mammals stimulate R _a NEFA to support exercise or shivering. In contrast, results reveal that the R _a NEFA of the rat remains constant during cold exposure (∼55 μmol kg⁻¹ min⁻¹). No preferential use of mono-unsaturated over saturated fatty acids could be demonstrated. The rat decreases its rate of fatty acid re-esterification from 48.4 ± 6.4 to 19.6 ± 6.3 μmol kg⁻¹ min⁻¹ to provide energy to shivering muscles. This study is the first to show that mammals do not only increase fatty acid availability for oxidation by stimulating R _a NEFA. Reallocation of fatty acids from re-esterification to oxidation is a novel, alternative strategy used by the rat to support shivering.     

Insulin and contraction increase nutritive blood flow in rat muscle in vivo determined by microdialysis of l-[14C]glucose

In the present study, a mathematical model using the microdialysis outflow: inflow (O/I) ratio of the novel analogue l -[¹⁴C]glucose has been developed which allows the calculation of the nutritive (and non-nutritive) flow in muscle as a proportion of total blood flow. Anaesthetized rats had microdialysis probes carrying l -[¹⁴C]glucose inserted through a calf muscle group (tibialis/plantaris/gastrocnemius). The nutritive fraction of total blood flow was determined under basal conditions and in response to contraction (electrical field stimulation), insulin (hyperinsulinaemic euglycaemic clamp with 10 mU min⁻¹ kg⁻¹ insulin) or saline control from limb blood flow and the microdialysis O/I ratio of l -[¹⁴C]glucose. Both contraction and insulin infusion decreased the O/I ratio of l -[¹⁴C]glucose and increased total limb blood flow. Calculations based on mathematical models using l -[¹⁴C]glucose O/I and limb blood flow revealed that during basal conditions, the nutritive fraction of total flow was 0.38 ± 0.06, indicating that basal flow was predominantly non-nutritive. Contraction and insulin increased the nutritive fraction to 0.82 ± 0.24 ( P < 0.05) and 0.52 ± 0.12 ( P < 0.05). Thus the increase in limb blood flow from insulin was fully accommodated by nutritive flow, while contraction increased nutritive flow at the expense of non-nutritive flow. This novel method using microdialysis and the O/I ratio of l -[¹⁴C]glucose allows the determination of the nutritive fraction of total flow in muscle as well as the proportion of total flow that may be redistributed in response to contraction and insulin.     

Cerebral non-oxidative carbohydrate consumption in humans driven by adrenaline

During brain activation, the decrease in the ratio between cerebral oxygen and carbohydrate uptake (6 O₂/(glucose +  ¹/₂  lactate); the oxygen–carbohydrate index, OCI) is attenuated by the non-selective β-adrenergic receptor antagonist propranolol, whereas OCI remains unaffected by the β₁-adrenergic receptor antagonist metroprolol. These observations suggest involvement of a β₂-adrenergic mechanism in non-oxidative metabolism for the brain. Therefore, we evaluated the effect of adrenaline (0.08 μg kg⁻¹ min⁻¹ i.v. for 15 min) and noradrenaline (0.5, 0.1 and 0.15 μg kg⁻¹ min⁻¹ i.v. for 20 min) on the arterial to internal jugular venous concentration differences (a-v diff) of O₂, glucose and lactate in healthy humans. Adrenaline ( n = 10) increased the arterial concentrations of O₂, glucose and lactate ( P < 0.05) and also increased the a-v diff for glucose from 0.6 ± 0.1 to 0.8 ± 0.2 m m (mean ± s.d. ; P < 0.05). The a-v diff for lactate shifted from a net cerebral release to an uptake and OCI was lowered from 5.1 ± 1.5 to 3.6 ± 0.4 ( P < 0.05) indicating an 8-fold increase in the rate of non-oxidative carbohydrate uptake during adrenaline infusion ( P < 0.01). Conversely, noradrenaline ( n = 8) did not affect the OCI despite an increase in the a-v diff for glucose ( P < 0.05). These results support that non-oxidative carbohydrate consumption for the brain is driven by a β₂-adrenergic mechanism, giving neurons an abundant provision of energy when plasma adrenaline increases.     

Long-term effects of streptozotocin-induced diabetes on the electrocardiogram, physical activity and body temperature in rats

In vivo biotelemetry studies have demonstrated that short-term streptozotocin (STZ)-induced diabetes is associated with a reduction in heart rate (HR) and heart rate variability (HRV) and prolongation of QT and QRS intervals. This study investigates the long-term effects of STZ-induced diabetes on the electrocardiogram (ECG), physical activity and body temperature. Transmitter devices were surgically implanted in the peritoneal cavity of young adult male Wistar rats. Electrodes from the transmitter were arranged in Einthoven bipolar lead II configuration. ECG, physical activity and body temperature data were continuously recorded with a telemetry system before and following the administration of STZ (60 mg kg⁻¹) for a period of 22 weeks. HR, physical activity and body temperature declined rapidly 3–5 days after the administration of STZ. The effects became conspicuous with time reaching a new steady state approximately 1–2 weeks after STZ treatment. HR at 4 weeks was 268 ± 5 beats min⁻¹ in diabetic rats compared to 347 ± 12 beats min⁻¹ in age-matched controls. HRV at 4 weeks was also significantly reduced after STZ treatment (18 ± 3 beats min⁻¹) compared to controls (33 ± 3 beats min⁻¹). HR and HRV were not additionally altered in either diabetic rats (266 ± 5 and 20 ± 4 beats min⁻¹) or age-matched controls (316 ± 6 and 25 ± 4 beats min⁻¹) at 22 weeks. Reduced physical activity and/or body temperature may partly underlie the reductions in HR and HRV. In addition, the increased power spectral low frequency/high frequency ratio from 4 weeks after STZ treatment may indicate an accompanying disturbance in sympathovagal balance.     

Economy of locomotion in high-altitude Tibetan migrants exposed to normoxia

High-altitude Tibetans undergo a pattern of adaptations to chronic hypoxia characterized, among others, by a more efficient aerobic performance compared with acclimatized lowlanders. To test whether such changes may persist upon descent to moderate altitude, oxygen uptake of 17 male Tibetan natives lifelong residents at 3500–4500 m was assessed within 1 month upon migration to 1300 m. Exercise protocols were: 5 min treadmill walking at 6 km h⁻¹ on increasing inclines from +5 to +15% and 5 min running at 10 km h⁻¹ on a +5% grade. The data (mean ± s.e.m. ) were compared with those obtained on Nepali lowlanders. When walking on +10, +12.5 and +15% inclines, net     of Tibetans was 25.2 ± 0.7, 29.1 ± 1.1 and 31.3 ± 0.9 ml kg⁻¹ min⁻¹, respectively, i.e. 8, 10 and 13% less ( P < 0.05) than that of Nepali. At the end of the heaviest load, blood lactate concentration was lower in Tibetans than in Nepali (6.0 ± 0.9 versus 8.9 ± 0.6 m m ; P < 0.05) . During running,     of Tibetans was 35.1 ± 0.8 versus 39.3 ± 0.7 ml kg⁻¹ min⁻¹ (i.e. 11% less; P < 0.01). In conclusion, during submaximal walking and running at 1300 m, Tibetans are still characterized by lower aerobic energy expenditure than control subjects that is not accounted for by differences in mechanical power output and/or compensated for by anaerobic glycolysis. These findings indicate that chronic hypoxia induces metabolic adaptations whose underlying mechanisms still need to be elucidated, that persist for at least 1 month upon descent to moderate altitude.     

Evidence of porcine and human endothelium activation by cancer-associated carbohydrates expressed on glycoproteins and tumour cells

It is well established that after metastatic cancer cells escape the primary tumour and enter the circulation, their interactions with microvascular endothelium of a target organ constitute an essential rate-limiting step in haematogenous cancer metastasis. However, the physiological and biochemical processes supporting neoplastic cell arrest and retention in the microcirculation are still poorly understood. In this study, we present experimental evidence that microvascular endothelium of metastasis-prone tissues undergoes activation in response to desialylated cancer-associated carbohydrate structures such as Thomsen–Friedenreich (TF) antigen (Galβ1–3GalNAc) expressed on circulating glycoproteins and neoplastic cells. The metastasis-associated endothelium activation, manifested by marked increase in endothelial cell surface galectin-3 expression, causes gradual decrease in cancer cell velocities (from 72 × 10²± 33 × 10²μm s⁻¹ to 7.6 × 10²± 1.9 × 10²μm s⁻¹, mean ± s.d. ) accompanied by a corresponding increase in the percentage of rolling cells (from 3.3%± 1.2% to 24.3%± 3.6%, mean ± s.d. ), and results in human breast and prostate carcinoma cell arrest and retention in the microvasculature. This process, which could be of high importance in haematogenous cancer metastasis, was inhibited efficiently by an anti-TF antigen function-blocking antibody. Carbohydrate-mediated endothelial activation could be a process of physiological significance as it probably occurs in the interactions between a variety of circulating constituents and the vessel wall.     

Protein synthesis rates in human muscles: neither anatomical location nor fibre-type composition are major determinants

In many animals the rate of protein synthesis is higher in slow-twitch, oxidative than fast-twitch, glycolytic muscles. To discover if muscles in the human body also show such differences, we measured [¹³C]leucine incorporation into proteins of anatomically distinct muscles of markedly different fibre-type composition (vastus lateralis, triceps, soleus) after an overnight fast and during infusion of a mixed amino acid solution (75 mg amino acids kg⁻¹ h⁻¹) in nine healthy, young men. Type-1 fibres contributed 83 ± 4% (mean ± s.e.m. ) of total fibres in soleus, 59 ± 3% in vastus lateralis and 22 ± 2% in triceps. The basal myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR, % h⁻¹) were 0.034 ± 0.001 and 0.064 ± 0.001 (soleus), 0.031 ± 0.001 and 0.060 ± 0.001 (vastus), and 0.027 ± 0.001 and 0.055 ± 0.001 (triceps). During amino acid infusion, myofibrillar protein FSR increased to 3-fold, and sarcoplasmic to 2-fold basal values ( P < 0.001). The differences between muscles, although significant statistically (triceps versus soleus and vastus lateralis, P < 0.05), were within ∼15%, biologically probably insignificant. The rates of collagen synthesis were not affected by amino acid infusion and varied by < 5% between muscles and experimental conditions.     

Mechanisms of acute natriuresis in normal humans on low sodium diet

This study evaluates the relative importance of several mechanisms possibly involved in the natriuresis elicited by slow sodium loading, i.e. the renin-angiotensin-aldosterone system (RAAS), mean arterial blood pressure (MAP), glomerular filtration rate (GFR), atrial natriuretic peptide (ANP), oxytocin and nitric oxide (NO). Eight seated subjects on standardised sodium intake (30 mmol NaCl day⁻¹) received isotonic saline intravenously (NaLoading: 20 μmol Na⁺ kg⁻¹ min⁻¹ or ≈11 ml min⁻¹ for 240 min). NaLoading did not change MAP or GFR (by clearance of ⁵¹Cr-EDTA). Significant natriuresis occurred within 1 h (from 9 ± 3 to 13 ± 2 μmol min⁻¹). A 6-fold increase was found during the last hour of infusion as plasma renin activity, angiotensin II (ANGII) and aldosterone decreased markedly. Sodium excretion continued to increase after NaLoading. During NaLoading, plasma renin activity and ANGII were linearly related ( R = 0.997) as were ANGII and aldosterone ( R = 0.999). The slopes were 0.40 p m ANGII (mi.u. renin activity)⁻¹ and 22 p m aldosterone (p m ANGII)⁻¹. Plasma ANP and oxytocin remained unchanged, as did the urinary excretion rates of cGMP and NO metabolites (NO_x). In conclusion, sodium excretion may increase 7-fold without changes in MAP, GFR, plasma ANP, plasma oxytocin, and cGMP- and NO_x excretion, but concomitant with marked decreases in circulating RAAS components. The immediate renal response to sodium excess appears to be fading of ANGII-mediated tubular sodium reabsorption. Subsequently the decrease in aldosterone may become important.     

Tetrahydrobiopterin augments endothelium-dependent dilatation in sedentary but not in habitually exercising older adults

Endothelium-dependent dilatation (EDD) is impaired with ageing in sedentary, but not in regularly exercising adults. We tested the hypotheses that differences in tetrahydrobiopterin (BH₄) bioactivity are key mechanisms explaining the impairment in EDD with sedentary ageing, and the maintenance of EDD with ageing in regularly exercising adults. Brachial artery flow-mediated dilatation (FMD), normalized for local shear stress, was measured after acute oral placebo or BH₄ in young sedentary (YS) ( n = 10; 22 ± 1 years, mean ± s.e.m. ), older sedentary (OS) ( n = 9; 62 ± 2), and older habitually aerobically trained (OT) ( n = 12; 66 ± 1) healthy men. At baseline, FMD was ∼50% lower in OS versus YS (1.12 ± 0.09 versus 0.57 ± 0.09 (Δmm (dyn cm⁻²)) × 10⁻², P < 0.001; 1 dyn = 10⁻⁵ N), but was preserved in OT (0.93 ± 0.08 (Δmm (dyn cm⁻²)) × 10⁻²). BH₄ administration improved FMD by ∼45% in OS (1.00 ± 0.10 (Δmm (dyn cm⁻²)) × 10⁻², P < 0.01 versus baseline), but did not affect FMD in YS or OT. Endothelium-independent dilatation neither differed between groups at baseline nor changed with BH₄ administration. These results suggest that BH₄ bioactivity may be a key mechanism involved in the impairment of conduit artery EDD with sedentary ageing, and the EDD-preserving effect of habitual exercise.     

Kinetic Analysis of Subset Growth and B-Cell Activation in Pokeweed Mitogen-Stimulated Cultures of Peripheral Blood Mononuclear Cells Depleted of or Enriched for OKT8+ Cells

Peripheral blood mononuclcar cells (PBMN) that were depleted of OKT8⁺ cells and stimulated with pokeweed mitogen (PWM) produced higher cell yields and higher numbers of plaque-forming cells than unfractionated PBMN. Conversely, OKT8-enriched PBMN, prepared by mixing unfractionated and OKT8⁺ cells in a ratio of 3:1, gave reduced cell growth and B-cell activation. In OKT8-depleted cultures, B cells, OKT4⁺ cells, OKT8⁺ cells, and OKM1⁺ cells increased in number between days 4 and 7 of culture by factors of 9.8, 5.9, 20.1, and 5.6 respectively, whereas growth rates for these subsets were 2.4, 1.0, 2.0, and 1.3 in unfractionated cultures and 0.9, 1.0, 1.2, and 0.6 in cultures enriched for OKT8⁺ cells. On day 7 of culture, 73±10% of B cells secreted immunoglobulin in unfractionated cultures, whereas only 21±10% of B cells were activated in OKT8-enriched cultures. Surprisingly, PWM stimulation of OKT8-depleted PBMN produced only 40±12% activated B cells.     

Purification of Human Interferon by Antibody Affinity Chromatography, using Highly Absorbed Anti-interferon

Antibodies against partially purified human leucocyte interferon (PIF) were bound to Sepharose 4B and crude interferons applied on this affinity column were purified, up to 8 × 10⁵ interferon units (IFU) per mg protein in one step. Antibodies against PIF were absorbed with immobilized crude human leucocyte interferon bound to Sepharose, whereby antibodies against impurities were predominantly removed. Extensively absorbed antisera were coupled to Sepharose and used for antibody affinity chromatography of crude interferon preparations. Leucocyte and fibroblast interferons were purified in one step with around 100% recovery, up to 1 × 10⁸ IFU per mg protein, and Namalva interferon up to 2 × 10⁷ IFU/mg. SDS electrophoresis of affinity-purified leucocyte interferon revealed that the interferon activity appeared in two bands (19,000 and 23,000 D).