Chronic K-supplementation decreases myocardial [Na,K-ATPase] and net K-uptake capacity in rodents

The effects of high K intake on plasma K, myocardial K content and Na,K-ATPase concentration and on myocardial K uptake during KCl infusion were evaluated in rodents. Myocardial Na,K-ATPase was quantified in crude homogenates by K-dependent pNPPase activity in rats, and in intact samples by3H-ouabain binding in guinea pigs. Na, K-ATPase alpha isoform distribution was assessed by immunoblotting. Plasma K was monitored in anesthetized rats during intravenous infusion of 0.75 mmol KCl/100 g body weight/h. A significant increase in plasma K was observed after 2 days of K supplementation, 4.9+/-0.2 (mean+/-s.e.m.)v 3.0+/-0.2 mmol/l in weight matched controls ( P<0.01,n=5) and this difference remained stable. After 1 day, a significant myocardial K content increase was obtained, 86. 2+/-3.0v 76.7+/-1.9 micromol/g wet weight (P<0.05, n=5); after 4 days myocardial K stabilized 4.9+/-1.2 micromol/g wet weight above control level (P<0.05,n=5). From the 4th day, a significant decrease in myocardial K-dependent pNPPase activity was observed, 1.18+/-0.04v 1. 31+/-0.01 micromol/min/g wet weight in weight matched controls (P<0. 05,n=5); after 2 weeks the decrease was 29% (P<0.05,n=5), with a reduction in alpha1-isoform abundance by 24% (P<0.05,n=5), and a tendency to a decrease in alpha2 of 10% (n.s.,n=5). The measurements were validated by 3H-ouabain binding to myocardial samples from guinea pigs K-supplemented for 2 weeks, showing a decrease of 21% (P<0.05,n=5). During KCl infusion, the myocardial K content increase rate was reduced by 52% (P<0.05) in the K-supplemented rats. The observed effects of K-supplementation on plasma K, myocardial K content and myocardial K-dependent pNPPase activity were abolished within 2 days after reallocation to chow with normal K content. In conclusion, high K-intake is associated with significantly and reversible increased plasma and myocardial K content, and decreased myocardial Na,K-ATPase concentration and net myocardial K uptake capacity. Thus, the heart is protected from major increases in intracellular K concentrations during chronically-high K-intake.     

Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.     

Erythropoietin-receptor expression and function during the initiation of murine yolk sac erythropoiesis

Although erythropoietin is necessary for definitive (fetal liver and bone marrow) erythropoiesis, the role of erythropoietin signaling in primitive (yolk sac) hematopoiesis has not been well defined. In situ hybridization studies have revealed that erythropoietin-receptor (EPOR) mRNA accumulation begins in mesoderm cell masses of the developing yolk sac of the neural plate stage embryo (E7.5) before the development of morphologically recognizable erythroblasts. EPOR mRNA is also present in yolk sac blood islands at early somite stages (E8.5). These findings suggest that EPOR functions during early stages of yolk sac erythropoiesis. We have used a serum-free murine yolk sac explant system (Palis et al., Blood 86:156, 1995) to investigate the initial differentiation of primitive erythroblasts from extraembryonic mesoderm cells. Exogenous erythropoietin increased both erythroblast numbers and betaH1-globin accumulation in yolk sac explants, suggesting that primary yolk sac erythroblasts are directly responsive to erythropoietin. An antisense oligodeoxynucleotide (ODN) experimental approach was used to examine the functional role of erythropoietin signaling during the initiation of yolk sac hematopoiesis in yolk sac explants. Antisense EPOR ODN produced a >50% reduction (p < 0.005) in the number of differentiating primitive erythroblasts, >95% reduction in betaH1-globin accumulation (p < 0.001), and a >50% reduction (p < 0.01) in the number of CFU-E and BFU-E compared with missense EPOR ODN-treated and untreated control explants. Antisense EPOR ODN also blocked the increase in primitive erythroblast number induced by exogenous erythropoietin. We conclude that erythropoietin/EPOR signaling is functionally active during the initial proliferation and differentiation of primary yolk sac erythroblasts. These results also suggest that other growth factor signaling cascades are active during the onset of mammalian erythropoiesis.     

Sustained left ansae subclaviae stimulation for a 5-hour period inhibits cesium-induced ventricular arrhythmogenesis in rabbits

The acute stimulation of the left ansae subclaviae (LAS) augments cesium chloride (Cs)- induced early afterdepolarizations (EADs) and ventricular arrhythmias. However, the effect of sustained cardiac sympathetic nerve stimulation on Cs-induced arrhythmogenesis remains to be elucidated. Three protocols were used. In protocol A, seven rabbits received continuous LAS stimulation for 5 h (study group), and were then given bolus injections of Cs at 5-min intervals (initial dose 1.5 mmol/kg, followed by repetitive doses 1 mmol/kg). Another eight rabbits received the same dose of Cs after a 5-h observation period without LAS stimulation (control group). The effects of 2 h of LAS stimulation were also assessed in five rabbits of each group. In protocol B, a bolus injection of Cs (1 mmol/kg) was administered during atrial pacing (cycle length: 250 ms) after a 5-h period with (study group, n = 5) or without (control group, n = 5) LAS stimulation, and the amplitude of Cs-induced EADs was measured. In protocol C, a bolus injection of isoproterenol (0.05 mg/kg) was given after a 5-h period with (study group, n = 5) or without (control group, n = 5) LAS stimulation to assess the sensitivity to beta-adrenergic stimulation. In protocol A, the cumulative doses of Cs required for the induction of ventricular tachycardia (VT) and fibrillation (VF) were significantly greater in the study group than in the control group (4.4 +/- 0.1 v 2.8 +/- 0.3 mmol/kg for VT, P<0.05; 5.8 +/- 0.3 v 4.1 +/- 0.4 mmol/kg for VF, P<0.05). However, 2 h of LAS stimulation did not influence the cumulative doses of Cs required for the induction of VT and VF. In protocol B, the amplitude of Cs-induced EADs was significantly lower in the study group than in the control group. In protocol C, the increase in rate-pressure product by isoproterenol was significantly less in study than in control groups (3492 +/- 612 v 6504 +/- 829, P<0.05). These results suggest that sustained LAS stimulation exerts a protective effect against Cs-induced arrhythmogenesis in the rabbit heart, which may be partially explained by desensitization to the beta-adrenergic stimulation.     

Endogenous erythropoietin in patients on regular dialysis therapy

In a group of 66 patients with chronic renal failure having regular dialyzation treatment the serum concentration of endogenous erythropoietin (EPO), haemoglobin levels (Hb), haematocrit (Ht) and serum creatinine (Cr) were assessed. The examined subjects were never treated with recombinant erythropoietin and deficiency of iron, folic acid and vitamin B12 was ruled out. Endogenous EPO was assessed by the authors own RIA method, normal values being 24-42 mU/ml. The mean EPO concentration in the whole group of patients was 37.4 +/- 15.3 mU/ml, whereby 12 patients had an EPO serum concentration higher than the upper range of normal values. Between EPO concentrations and Hb values a certain positive correlation was found (r = 0.42). A similar relationship was revealed also between EPO concentrations and Ht values (r = 0.41). Patients with EPO values higher than 42 mU/ml had, as compared with the other patients, significantly higher values of erythrocytes (p .001). Statistical analysis did not reveal any relationship between EPO and Cr concentrations (r = -0.04). A low negative correlation was found between Cr and Hb values (r = -0.31) and between Cr and Ht values (r = -0.25). In the discussion the authors analyze the contemporary state of the problem of anaemia in chronic renal failure. Based on hitherto assembled knowledge they formulated the hypothesis ascribing considerable pathogenetic importance in the development of anemia to reduced sensitivity of bone marrow to EPO, probably as a result of retention of uraemic toxins and inhibitors of erythropoiesis. Inadequate EPO formation could be only a factor which makes it impossible for the developing anaemia to compensate and is due to an animpaired feedback at the level of recognition of the hypoxic signal.     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Recombinant human erythropoietin resistance in hemodialysis. Effects of paired filtration dialysis

A percentage of hemodialysis (HD) patients are resistant to recombinant human erythropoietin (rHuEPO), a phenomenon that occurs less frequently in patients dialyzed with biocompatible membranes (M) and in peritoneal dialysis. The authors evaluated the effects of paired filtration dialysis (PFD)--a dialysis technique based on the use of an emophan M in conjunction with a polysulphone M--on erythropoiesis in HD patients resistant to rHuEPO. Twelve HD patients with anemia resistant to long-term therapy with rHuEPO (200.24 U/kg body weight three times per week intravenously for 10.2 months) were studied. Patients had been treated for an average of 46.9 months with bicarbonate HD, using cuprophan M (Phase A) and, successively, for 12 months by PFD (Phase B). The following parameters were evaluated monthly: 1) hemoglobin and hematocrit values; 2) serum levels of erythropoietin (EPO); and 3) serum levels of interleukin (IL)-3, IL-6, IL-10, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma). At the end of Phase A and Phase B, patients underwent bone marrow biopsies to evaluate 1) bone marrow burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) proliferative capacity, and 2) bone marrow mononuclear cell EPO-receptor (EPO-R) number. During Phase B, there was a progressive rise in hematocrit and hemoglobin values, so that within the sixth month, the rHuEPO dose was reduced to 80 +/- 15 U/kg body weight three times per week. At the same time, an increase in serum IL-3, IL-6, and IL-10 levels was seen, whereas serum IL-1 beta, TNF-alpha, and IFN-gamma levels decreased. This was accompanied by a rise in BFU-E and CFU-E growth and in bone marrow mononuclear cell EPO-R number. During Phase B, after the dialysis session, serum EPO levels increased by about 30% in comparison with pre dialysis values, whereas during Phase A they decreased by about 14%. In HD patients, EPO resistance may caused either by absorption of rHuEPO to the cuprophan M, or an increased release of cytokines that inhibit erythropoiesis, such as IL-1 beta, TNF-alpha, and IFN-gamma, and to a decrease in stimulatory cytokines such as IL-3, IL-6, and IL-10. These negative phenomena are reversed by the use of biocompatible dialysis techniques such as PFD.     

Role of nitric oxide in the regulation of the hepatic microcirculation in vivo

BACKGROUND/AIMS: Nitric oxide (NO) is an important mediator in the regulation of vascular tone. However, no data exist on the physiological role of NO in the regulation of the hepatic microcirculation. This study was designed to evaluate the role of NO in the hepatic microcirculation in vivo under physiological conditions. METHODS: The hepatic microcirculation was investigated in anesthetized rats by intravital fluorescence microscopy after injection of fluorescein-isothiocyanate-labeled erythrocytes. Following assessment of baseline sinusoidal perfusion, animals were randomly treated with L-NMMA (n=6), L-arginine (n=6), nitroprusside sodium (NPS, n=5) or a comparable volume of NaCl (n=4). Drugs were given through a portal vein catheter at three doses (Dx), each followed by intravital microscopy. L-NMMA was given: 5 mg/kg (D1), 25 mg/kg (D2), 50 mg/kg (D3); L-arginine 30 mg/kg (D1), 150 mg/kg (D2), 300 mg/kg (D3); and NPS continuously 80 microg x kg(-1) x h(-1). RESULTS: L-NMMA induced a significant increase of mean arterial blood pressure (MAP) (114 vs. 129 mm Hg; p<0.05). In contrast, MAP of NPS-treated animals decreased (107 vs. 91 mm Hg; p<0.01) whereas MAP of animals receiving L-arginine did not significantly differ. Sinusoidal blood flow revealed dose-dependent changes: L-NMMA significantly decreased perfusion of sinusoids (D1: 65%, D2: 57%, D3: 50% of baseline, p<0.05). Injection of L-arginine increased the sinusoidal flow even with the lowest dose (D1: 137%, D2: 133%, D3: 123%, p<0.05). Continuous infusion of NPS had little effect on sinusoidal blood flow at the first and second times of microscopy but sinusoidal blood flow was significantly increased at the third time (D1: 103%, D2: 106%, D3: 122%). CONCLUSIONS: Inhibition of NOS results in a dose-dependent disturbance of the hepatic microcirculation despite significantly increased MAP, whereas L-arginine increases the sinusoidal blood flow. The results indicate an important role for NO in the regulatory mechanisms of hepatic sinusoidal perfusion under physiological conditions.     

The study of molar furcation involvements in adult periodontitis. II. Age, sex, location and prevalence

The purposes of the study were as follows: (1) to evaluate the molar furcation involvement and number of molar correlated with age and sex; and (2) to study the relationship between the means of alveolar bone loss and associated factors of molar furcation involvements (FIs). 1102 molars (703 males and 399 females) were measured in 219 individuals (136 males and 83 females) for the alveolar bone loss and associated factors of molar furcation involvements. Based on the results, we conclude the following: (1) the higher prevalence of FI was in the mandibular first molar (94.6%), whereas the lowest prevalence of FI was in maxillary second molar; (2) except for the mandibular first molar, the prevalence of molar FI markedly increased with an increased age group (16 & 26, r = 0.335, p < 0.01; 17 & 27, r = 0.345, p < 0.01; 37 & 47, r = 0.239, p < 0.01); (3) the prevalence of molar FI was significantly higher in males than in females (p < 0.05); (4) the mean number of molar FI was significant greater in males (mean = 3.45) than in females (mean = 2.69); (5) factors such as age (r = 0.222, p < 0.01), sex, (r = 0.145, p < 0.05), number of remaining teeth (r = -0.330, p < 0.01) and molar FI (r = 0.471, p < 0.01) are strongly associated with the mean alveolar bone loss of molars.     

Radioimmunoguided surgery benefits in carcinoembryonic antigen-directed second-look surgery in the asymptomatic patient after curative resection of colorectal cancer

A daily ingestion of 1000 to 1500 mg elemental calcium associated with vitamin D supplement is presently considered to be the adequate and least expensive therapy for senile osteoporosis. There exists only scarce data about calcium absorption with available calcium salts in elderly patients. We have compared the digestive absorption of calcium (Ca) citrate in soluble and solid form and calcium gluconolactate-carbonate in 15 young and 20 elderly, healthy women using the oral calcium loading test. The subjects were divided into two groups. In the first group, the absorption of solid Ca citrate (1000 mg Ca element) was compared to the absorption of Ca gluconolactate-carbonate (1000 mg Ca element) both in young (n = 7) and elderly women (n = 10). In the second group, the absorption of soluble Ca citrate (1000 mg Ca element) was compared to the absorption of Ca gluconolactate-carbonate (1000 mg Ca element) in young (n = 8) and elderly (n = 10) women. In the preload phase, basal calciuria was increased in elderly women (p < 0.01) although basal calcemia was similar in young and elderly women. After oral administration of the calcium salts, an increase in plasma Ca was observed in both groups which was greater for soluble Ca citrate and Ca gluconolactate than for solid Ca citrate. In young women, the increase in plasma calcium was significantly higher with soluble Ca citrate compared to Ca gluconolactate (p < 0.05). In elderly women, the postload calciuria was significantly higher for soluble Ca citrate (p < 0.05) and Ca gluconolactate (p < 0.05) compared to solid Ca citrate. A similar pattern was observed in young women, although it was not significant. In conclusion, an oral load of 1000 mg soluble Ca citrate and Ca gluconolactate-carbonate induces significant biochemical changes suggesting a better digestive absorption compared to Ca citrate in solid form, both in young and elderly women. We did not observe different response, between young and old patients.     

Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01-1 microm) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05). 2. The mu-, delta- and kappa-opioid receptor antagonists, naloxone (0.3 microM), naltrindole (3 microM) and norbinaltorphimine (1 microm), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 microM; P>0.05). 3. The novel nociceptin antagonist, [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (0.03-1 microM); the sigma ligands, carbetapentane (30 microM), 3-phenylpiperidine (30-100 microM) and (+)-cyclazocine (10-100 microM) significantly reversed the inhibitory effect of nociceptin (0.03 microM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 microM) at any concentration tested (P>0.05). 4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 microM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59+11% (n=5) inhibition of EFS-induced release of SP-LI. 5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.     

Histological study of saphenous vein graft disease after coronary artery bypass grafting

This study examines whether platelet-activating factor (PAF) is involved in the occurrence of vasospasm after subarachnoid hemorrhage (SAH). A vasospasm model was produced in rabbits, with animals in six experimental groups receiving two subarachnoid injections of autologous arterial blood with the addition of one of the following; saline (Control Group 1), 25% dimethyl sulfoxide (Control Group 2), PAF (1, 2.5, 5, or 10 micrograms), CV6209 (10 or 100 micrograms), BN52021 (10 or 100 micrograms), or anti-PAF immunoglobulin G (IgG, 50 or 500 micrograms). No significant differences were detected between Control Groups 1 and 2 with regard to neurological deterioration and basilar artery constriction after SAH was induced. Administration of PAF together with autologous blood aggravated neurological deficits in a dose-dependent manner (r = 0.724, p < 0.001) and produced basilar artery constriction at two doses each of 2.5 micrograms (p < 0.05), 5 micrograms (p < 0.01), and 10 micrograms (p < 0.01). Neurological deterioration was prevented in rabbits receiving an intracisternal administration of either PAF antagonist CV6209 or BN52021 or anti-PAF IgG (p < 0.01 at a total dose of 20 micrograms and p < 0.05 at a total dose of 200 micrograms CV6209, p < 0.01 at total doses of 20 and 200 micrograms BN52021, and p < 0.01 at total doses of 100 and 1000 micrograms anti-PAF IgG). A reduction in basilar artery constriction was achieved by the injection of anti-PAF IgG (p < 0.05 at total doses of 100 and 1000 micrograms). Histological examination at autopsy on Days 14 to 21 showed mainly ischemic changes in the brain, including selective neuronal necrosis and cerebral infarction. The control and PAF groups showed marked ischemic changes. On the other hand, no ischemic changes were noted in the anti-PAF IgG group, and only 9% of animals in the CV6209 group and 25% in the BN52021 group demonstrated selective neuronal necrosis or infarction. This study thus provides evidence to support the role of PAF in the pathogenesis of vasospasm after SAH.     

Plasma levels of glutathione, alpha-tocopherol and lipid peroxides in polytraumatized patients; evidence for a stimulating effect of TNF alpha on glutathione synthesis

Prognosis and outcome of polytraumatized patients are determined by the possible development of multiple organ failure (MOF). Among the direct traumatic organ damage, it is caused by a systemic inflammatory reaction. This might be triggered by an activation of the inflammatory mediator cascade following hemorrhagic-traumatic shock as well as by oxygen-derived free radicals (ROS). The aim of our present study was to answer the following questions: 1. Is the "oxidative stress" measurable during the development of MOF after polytraumatic injury? 2. Is there a relation between the activation of the inflammatory mediator cascade and changes of the organism's antioxidative system? The study group included 26 patients (15 survivors, 11 non-survivors) suffering from severe polytraumatic injury (Hannover Polytrauma Score 12-63 points). Plasma levels of reduced (GSH) and oxidized (GSSG) glutathione alpha-tocopherol (TOC), lipid peroxides (expressed in terms of thiobarbituric acid reagible substances = TBARS), and tumor necrosis factor alpha (TNF) were measured each day from the point of admission on the ICU until the discharge or death of the patients. The following results were obtained: Independent from the outcome, we observed a continuous loss of plasma sulfhydryl groups and TOC. In the patients developing a MOF score > 5 on 10th day after injury (n = 6), a significant increase in plasma GSSG level was measurable. Additionally, a total loss of plasma GSH was seen in some of these patients indicating the collapse of the GSH-dependent antioxidative system. Similar changes were never observed in patients with MOF score < or = 5 on 10th day after injury (n = 15). In this group, a significant correlation between plasma TNF peaks and short time GSH boosts was obtained as a possible indicative for a stimulating effect of TNF on GSH synthesis. It can be concluded that processes of oxidative stress in connection with a consumption of endogenic antioxidants might be able to promote the development of MOF after polytraumatic injury.     

The effect of propanediol on the morphology of fresh and frozen equine embryos

Seventeen horse embryos recovered on the sixth day after spontaneous ovulation were; 1) washed in PBS (n = 6), 2) treated with 1.5 M 1-2 propanediol (n = 6) or, 3) frozen and thawed using 1.5 M propanediol as the cryoprotectant (n = 5). After treatment, the embryos were incubated for 6 h in medium before they were fixed, serially sectioned and examined microscopically to count the total numbers of interphase, mitotic and pycnotic nuclei. Significant differences were measured only in the mean proportions of pycnotic cells (+/- s.d.), both between the control (9.2 +/- 7.3%) and frozen-thawed embryos (52.8 +/- 37.1%; P<0.05) and between the propanediol-treated (10.8 +/- 4.6%) and the frozen-thawed embryos (P<0.01). Propanediol appears to be minimally toxic to equine embryos but it is a poor cryoprotectant.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

Increased formation of distinct F2 isoprostanes in hypercholesterolemia

BACKGROUND: F2 isoprostanes are stable, free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo. METHODS AND RESULTS: Specific assays were developed by use of mass spectrometry for the F2 isoprostanes iPF2alpha-III and iPF2alpha-VI and arachidonic acid (AA). Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF2alpha-III (pmol/mmol creatinine) was elevated (P<0.0005) in homozygous familial hypercholesterolemic (HFH) patients (85+/-5. 5; n=38) compared with age- and sex-matched normocholesterolemic control subjects (58+/-4.2; n=38), as were levels of iPF2alpha-VI (281+/-22 versus 175+/-13; P<0.0005). Serum cholesterol correlated with urinary iPF2alpha-III (r=0.41; P<0.02) and iPF2alpha-VI (r=0. 39; P<0.03) in HFH patients. Urinary excretion of iPF2alpha-III (81+/-10 versus 59+/-4; P<0.05) and iPF2alpha-VI (195+/-18 versus 149+/-20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n=24) compared with their controls. Urinary excretion of iPF2alpha-III and iPF2alpha-VI was correlated (r=0.57; P<0.0001; n=106). LDL iPF2alpha-III levels (ng/mg arachidonate) were elevated (P<0.01) in HFH patients (0.32+/-0.08) compared with controls (0.09+/-0.02). The concentrations of iPF2-III in LDL and urine were significantly correlated (r=0.42; P<0.05) in HFH patients. CONCLUSIONS: Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.     

In vivo effects of recombinant human erythropoietin on bone marrow hematopoiesis in patients with chronic renal failure

Studies of hemopoietic progenitors and precursors in bone marrow before and after two months of recombinant human erythropoietin (rhEpo) therapy in 12 patients with uremic anemia are the subject of this investigation caried out in order to have a better insight into the effect of Epo in vivo. Eight patients were on hemodialysis and four others were predialysis patients with chronic renal failure. The starting dose of rhEpo was 30-50 U/kg bw and was increased by 50 percent every four weeks. The mean hemoglobin values rose from 6.08 +/- 1.03 to 9.8 +/- 1.98 g/dl at the time of study. The number of bone marrow derived erythroid colonies, both early (BFU-E) and late (CFU-E) were found to be higher than subnormal values, found before the therapy. The percentage of erythroid progenitors in cell cycle increased to higher than normal values for BFU-E and to normal values for CFU-E. At the same time granulocytic progenitors (CFU-GM) decreased to the range of normal values (67.3 per 10 superset5 cells). Slightly increased Epo levels (approx. 30mU/ml) during the replacement therapy were optimal for correction of anemia. The rhEpo therapy induced an increase of percentage of erythroblasts and the decrease of myeloid to erythroid ratio (M/E) in the bone marrow. Only in predialysis patients in whom the target hemoglobin values were achieved by rhEpo therapy at the time of the study the percentage of erythroblasts in the bone marrow increased to normal values. Increase of erythroblasts in bone marrow in patients under two months of substitutive therapy with rhEpo with the increase of both, early and late erythroid progenitors we have observed, is significant indicating the stimulative effect of rhEpo on all subsets of erythropoiesis leading to normalization of erythropoiesis at all levels. No stimulative effect of rhEpo replacement therapy on granulopoiesis was observed.     

Granulocyte colony-stimulating factor (G-CSF) dose-dependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF

For 10 consecutive patients in our unit who did not show a significant rise in blood progenitor cells within 14 days following chemotherapy and G-CSF, we increased the G-CSF dose from 5 to 10 microg/kg/day (n = 9) or from 10 to 15 microg/kg/day (n = 1). As a result, there were significant increases in total yield as well as yield per apheresis of mononuclear cells, CD34+ cells and CFU-GM (P < 0.025, <0.01 and <0.005, respectively). After G-CSF dose escalation, six of the 10 patients had sufficient CD34+ cells for performing transplantation. These results demonstrate a dose-dependent response of progenitor cell mobilization by G-CSF when used in combination with chemotherapy. Moreover, increasing the dose of G-CSF as late as the third week of mobilization may still provide sufficient cell yield even with patients who did not show a significant mobilization with conventional doses of G-CSF.     

Urinary excretion of free and acetylated polyamines in hepatocellular carcinoma

Bone mineral density (BMD), and associated biochemical and endocrine markers were compared in a group of runners with menstrual dysfunction (IR, n=13), and a group of performance matched eumenorrheic runners (R, n=15). All subjects claimed to have normal eating habits. Body height and weight, body mass index, and amount of body fat were similar. The IR group consisted of 5 presently oligomenorrheic (O) and 8 presently amenorrheic (A) runners. The BMD values of the athletes were additionally compared with corresponding values in a reference group (C) of healthy age matched controls (n=54). BMD values were significantly lower in IR compared with R on all measuring sites: Total body (-9%, p=0.03), femoral neck (-11%, p=0.01), lumbar spine (-12%, p=0.001), lower leg (-6.5%, p=0.03) and arms (-7%, p=0.01). In addition, IR athletes had lower total body (-5%, p=0.01), and lumbar spine BMD (-10%, p=0.001) than C. No differences were observed in serum IGF-1, SHBG, testosterone and cortisol, or in the biochemical marker of bone formation (osteocalcin) and bone resorption (1 CTP). Values of serum E2, FSH and LH were low in IR and normal in R. TSH was in the normal range in both groups, but f-T4 was significantly lower in IR than in R. The athletes were furthermore grouped according to past and present menstrual dysfunction severity. At all measuring sites, with the exception of the lower leg, increasing menstrual dysfunction severity was linearly associated with declining BMD values (p<0.05). In conclusion, even highly conditioned cortical bone tissue seems to be negatively related to menstrual disorders, which may serve to explain the high incidence of stress fractures in athletes with menstrual disorders. Single measurements of biochemical markers of bone resorption and formation may not reflect the current bone status.