Gene expression of LDL receptor, HMG-CoA reductase, and cholesterol-7 alpha-hydroxylase in chronic renal failure

BACKGROUND: Chronic renal failure (CRF) is associated with an atherogenic lipid profile and an increased risk of ischaemic cardiovascular disease. The associated hyperlipidaemia is reportedly ameliorated by erythropoietin (Epo) therapy. According to a recent report, rats studied 3 weeks after 5/6 nephrectomy and fed a high- protein diet exhibited increased activities of hepatic HMG-CoA reductase (HMG-CoAR) and cholesterol 7 alpha-hydroxylase (Ch-7 alpha- H), despite normal corresponding mRNA values. DESIGN AND METHODS: This study was designed to examine the effects of naturally progressing CRF of longer duration as well as those of Epo therapy on gene expressions of the key factors involved in hepatic cholesterol metabolism, i.e., LDL receptor (LDLR), HMG-CoAR, and Ch-7 alpha-H. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy), Epo-treated CRF group (given Epo 150 U/kg/twice weekly) and sham-operated, placebo- treated normal controls. They were allowed free access to regular rat chow and studied 6 weeks after surgery. Liver mRNAs and protein mass or activities of the above factors were studied. RESULTS: Plasma cholesterol concentration was significantly increased in the CRF group (P < 0.001) and was modestly lowered (P < 0.05) by Epo therapy. However, microsomal cholesterol concentration and LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA as well as HMG-CoAR activity, and Ch-7 alpha-H and LDLR protein mass measurements were virtually identical in the three groups. Thus, hepatic LDLR, HMG-CoAR, and Ch-7 alpha-H mRNA and protein measurements in rats with CRF were similar to those of the normal control group representing an inappropriate response to the associated hypercholesterolemia. Epo therapy led to partial amelioration of CRF- associated hypercholesterolaemia with no discernible effect on hepatic tissue expression of the above factors.      

Impaired expression of glycoproteins on resting and stimulated platelets in uraemic patients.

BACKGROUND: The increased bleeding tendency of chronic renal failure (CRF) patients has been attributed to platelet dysfunction. However, reports on various platelet functions in uraemic patients have been conflicting. The present study sought to analyse platelet function by examining their surface glycoproteins in well-identified populations of CRF patients. METHODS: Three groups were studied: 22 chronic haemodialysis (HD) patients, 25 conservatively treated patients with CRF and 30 controls. Bleeding tendency was assessed by measuring bleeding time and by recording current haemorrhagic symptoms. We measured the fibrinogen receptor GPIIbIIIa, the von Willebrand receptor GPIb, and P-selectin levels on the platelet surface using flow cytometry. RESULTS: Forty percent of CRF and 45% of HD patients had bleeding. The bleeding time was similar in the HD and CRF groups, but was longer in both groups than in controls. In resting platelets, GPIb expression was lower in CRF patients than in controls. In stimulated platelets (i) GPIb expression was higher in HD patients than in both controls and CRF patients; and (ii) GPIIbIIIa and P-selectin expression were lower in CRF and HD patients than in controls. CONCLUSIONS: These findings indicate that uraemic platelets are hyporesponsive to stimulation.     

Erythropoietin in patients with acute renal failure and continuous veno-venous haemofiltration

Erythropoietin (Epo) is a glycoprotein hormone produced in the kidney in response to hypoxia or anaemia. In acute renal failure (ARF) anaemia also occurs and current opinion is that Epo production is depressed with inappropriately low plasma levels throughout the uraemic phase. Our study was designed to determine the excretion of Epo in patients with ARF. Fifty-nine ventilated patients were studied, 39 with ARF and continuous veno-venous haemofiltration therapy (group 1) and 13 patients with normal renal function who served as a control group (group 2). All patients with ARF were anaemic and needed a mean transfusion of 0.6 units/day. Values for vitamin B12, folic acid, serum iron and ferritin were normal. While patients with normal renal function had Epo values within the normal range, patients with ARF had significantly higher values at the onset of haemofiltration therapy. Mean Epo (mean±SEM) values on days 0–2 were 92.6±11.7 mU/ml in group 1 and 16.5±6.4 mU/ml in group 2 (p<0.0002). Epo levels declined in group 1 to 49±10.5 mU/ml on days 9 and 10 compared to 23±9.1 mU/ml in group 2 (ns). These values were maintained until the end of the observation period. No differences were seen between oliguric and non-oliguric patients. Our data show that patients with ARF have increased Epo levels at the beginning of the disease with a strong tendency to decrease, suggesting that there might be inadequate Epo levels during the course of acute renal failure.     

Erythropoietin enhances recovery after cisplatin-induced acute renal failure in the rat.

BACKGROUND: Erythropoietin (Epo) is a growth factor whose synthesis mainly takes place in the kidney. Epo has been shown to support the growth not only of erythroid progenitor cells but also of certain other cell types. We attempted to establish whether Epo enhances the recovery from acute renal failure induced by cisplatin. METHODS: Sprague-Dawley rats were randomized into three groups. In the cisplatin group, animals received one intraperitoneal injection of cisplatin (6 mg/kg) and a daily injection of placebo for 9 days. In the cisplatin+Epo group, animals received intrapertoneal cisplatin and a daily injection of Epo (100 IU/kg) for 9 days. In the control group, animals received both placebo preparations alone. Para-aminohippuric acid and inulin clearances were determined after 4 and 9 days to evaluate renal blood flow and glomerular filtration rate. In addition, light microscopy and immunohistochemistry examinations were performed, and in situ proliferating cell nuclear antigen (PCNA) staining was done to estimate the degree of renal tubular cell regenerative activity. The potential role of epithelial growth factor (EGF) was evaluated by semi-quantitative assessment of EGF immunostaining. RESULTS: Renal blood flow and glomerular filtration rate decreased significantly in cisplatin and cisplatin+Epo groups versus control group at day 4. However, at day 9, they both were significantly greater in cisplatin+Epo-treated animals than in rats that had received cisplatin alone. Tubular cell regeneration was significantly enhanced at day 4 in cisplatin+Epo group, compared with cisplatin and control groups respectively. EGF immunostaining was not significantly different between the three groups. CONCLUSION: Epo significantly enhanced the rate of recovery from acute renal failure induced by cisplatin. PCNA staining indicated that Epo might act directly via stimulation of tubular cell regeneration.     

Changes of neuronal calcium channel following brain damage induced by injection of pertussin bacilli in rats

OBJECTIVE: To explore changes of neuronal calcium channel following brain damage induced by injection of pertussis bacilli in rats, and to investigate the relationship between cytosolic free calcium concentration ([Ca(2+)](i)) in the synaptosome and Ca(2+)-ATPase activities of mitochondria. METHODS: The level of [Ca(2+)](i) in the synaptosome and Ca(2+)-ATPase activities of mitochondria in the acute brain damage induced by injection of pertussis bacilli (PB) in rat was determined and nimodipine was administrated to show its effects on [Ca(2+)](i) in the synaptosome and on alteration of Ca(2+)-ATPase activity in the mitochondria. Seventy-three rats were randomly divided into four groups, ie, normal control group (Group A), sham-operation control group (Group B), PB group (Group C) and nimodipine treatment group (Group D). RESULTS: The level of [Ca(2+)](i) was significantly increased in the PB-injected cerebral hemisphere in the Group C as compared with that in the Group A and the Group B at 30 minutes after injection of PB. The level of [Ca(2+)](i) was kept higher in the 4 hours and 24 hours subgroups after the injection in the Group C (P<0.05). In contrast, the Ca(2+)-ATPase activities were decreased remarkably among all of the subgroups in the Group C. Nimodipine, which was administered after injection of PB, could significantly decrease the [Ca(2+)](i) and increase the activity of Ca(2+)-ATPase (P<0.05). CONCLUSIONS: The neuronal calcium channel is opened after injection of PB. There is a negative correlation between activities of Ca(2+)-ATPase and [Ca(2+)](i). Nimodipine can reduce brain damage through stimulating the activities of Ca(2+)-ATPase in the mitochondria, and decrease the level of [Ca(2+)](i) in the synaptosome. Treatment with nimodipine dramatically reduces the effects of brain damage induced by injection of PB.     

Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

BACKGROUND: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. METHODS: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. RESULTS: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. CONCLUSIONS: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.     

Erythropoietin reduces ischemia-reperfusion injury in the rat liver

BACKGROUND: Human recombinant erythropoietin (Epo) has recently been shown to be a potent protector of ischemic damage in various organ systems. A significant reduction of stroke injury following cerebral ischemia has been postulated as well as improved cardiomyocyte function after myocardial infarction in tissue pretreated with Epo. It was the aim of this study to evaluate the effects of Epo in liver ischemia. MATERIAL AND METHODS: Rats were subjected to 45 min of warm hepatic ischemia. Animals were either pretreated with 1,000 IU of Epo in three doses or received 1,000 IU into the portal vein 30 min before ischemia. Control animals received saline at the same time points before ischemia. Animals were than sacrificed 6, 12, 24, 48 h and 7 days after surgery and transaminases were measured. Liver specimens were evaluated regarding apoptosis, necrosis and regeneration capacity. RESULTS: Apoptosis rates were dramatically reduced in animals pretreated with Epo while mRNA of tumor necrosis factor-alpha and STAT-3 were upregulated in all groups. Intraportal venous injection displayed superiority to subcutaneous preconditioning. Transaminases were significantly reduced among the Epo-treated animals after 6 and 12 h. CONCLUSION: Our data suggests a protective effect of Epo in warm hepatic ischemia and reperfusion injury in the rat.     

Mechanism of estrogens-induced increases in intracellular Ca(2+) in PC3 human prostate cancer cells

BACKGROUND: The effect of estrogens (diethylstilbestrol [DES], 17 beta-estradiol) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in hormone-insensitive PC3 human prostate cancer cells was examined. METHODS: [Ca(2+)](i) changes in suspended cells were measured by using the Ca(2+)-sensitive fluorescent dye fura-2. RESULTS: Estrogens (1--20 microM) increased [Ca(2+)](i) concentration-dependently with DES being more potent. Ca(2+) removal inhibited 50 +/- 10% of the signal. In Ca(2+)-free medium, pretreatment with 20 microM estrogens abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), but pretreatment with CCCP and thapsigargin did not alter DES-induced Ca(2+) release and partly inhibited 17 beta-estradiol-induced Ca(2+) release. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 1- 20 microM estrogens in Ca(2+)-free medium. Pretreatment with 1 microM U73122 to block phospholipase C-coupled inositol 1,4,5-trisphosphate formation did not alter estrogens-induced Ca(2+) release. The effect of 20 microM estrogen on [Ca(2+)](i) was not affected by pretreatment with 0.1 microM estrogens. CONCLUSIONS: Estrogen induced significant Ca(2+) release and Ca(2+) influx in an inositol 1,4,5-trisphosphate-independent manner in PC3 cells. These effects of estrogens on Ca(2+) signaling appear to be nongenomic. Prostate 47:141-148, 2001.     

Intravenous iron supplementation for the treatment of anaemia in pre-dialyzed chronic renal failure patients.

BACKGROUND: Intravenous iron is a recognized therapy of anaemia in chronic haemodialyzed patients, especially in those receiving erythropoietin (Epo), while its role in the anaemia of pre-dialyzed chronic renal failure (CRF) patients is much less clear. This study attempted to evaluate the effects of intravenous iron in anaemic pre-dialyzed patients. METHODS: Sixty anaemic (haemoglobin<11 g/dl) non-diabetic patients with moderate CRF [32 males, 28 females; mean age 52.2+/-12.5 years; mean glomerular filtration rate 36.2+/-5.2 ml/min], without iron deficiency, iron overload or inflammation, without concomitant erythropoietin treatment and without any previous iron therapy were enrolled. Intravenous iron was administered as iron sucrose, 200 mg elemental iron per month for 12 months, with 1 month pre-study survey and 1 month follow-up after the last iron dose. RESULTS: Intravenous iron supplementation was associated with a significant increase in haemoglobin (from 9.7+/-1.1 at the baseline to 11.3+/-2.5 g/dl after 12 months, a mean increase of 1.6 g/dl), a further 36% of patients reaching the target haemoglobin of 10 g/dl. There was a significant increase in serum iron from 73.9+/-17.2 to 101.8+/-12.2 microg/dl, in serum ferritin from 98.0 (24.8-139.0) to 442.5 (86.0-496.0) microg/l and in transferrin saturation from 21.6+/-2.6 to 33.6+/-3.2%. No worsening of renal function, no increase in blood pressure and no other side effects were noted. CONCLUSIONS: Intravenous iron therapy in pre-dialysis patients with no Epo seems often to ameliorate the anaemia, avoiding the necessity of Epo or blood transfusions in one-third of pre-dialyzed non-diabetic patients. Intravenous iron supplementation appears to be an effective and safe treatment for anaemia in pre-dialysis CRF patients.     

Changes of myocardial mitochondrial Ca(2+) transport and mechanism in the early stage after severe burns

OBJECTIVE: To investigate the change of myocardial mitochondrial Ca(2+) transport and its mechanism in the early stage after burns. METHODS: Forty-eight Wistar rats were randomized into a normal control group (n=8) and a burns group (n=40). The rats of the burns group were given a 30%TBSA full-thickness. Myocardial mitochondria were isolated from normal and scalded rats which were sacrificed at the 1st, 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca(2+) transport velocity, membrane potential (MP), ATP content and cytosolic Ca(2+) concentration [Ca(2+)](c) were determined. The effects of exogenous ATP on mitochondrial Ca(2+) transport velocity were also investigated. RESULTS: Mitochondrial Ca(2+) uptake velocity of the 1st hour post-burn was higher than that of the control, and Ca(2+) release velocity did not change significantly, but mitochondrial Ca(2+) transport velocity, MP and ATP content were all decreased at the 3rd, 6th, 12th and 24th hour post-burn. Mitochondrial Ca(2+) uptake velocity was positively correlated with MP after burn, and Ca(2+) release velocity with mitochondrial ATP content. [Ca(2+)](c) was increased at the 3rd, 6th, 12th and 24th hour post-burn. Exogenous ATP increased myocardial mitochondrial Ca(2+) uptake velocity of rats at the 3rd and 6th hour post-burn and Ca(2+) release velocity at the 3rd, 6th and 12th hour post-burn. CONCLUSIONS: Increase of [Ca(2+)](c) led to reinforcement of mitochondrial Ca(2+) uptake at the beginning of the post-burns period. ATP depletion and MP collapse cause myocardial mitochondrial Ca(2+) transport disorder in the following stages.     

In situ imaging of the autonomous intracellular Ca(2+) oscillations of osteoblasts and osteocytes in bone

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca(2+) acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca(2+) dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca(2+) imaging system that allows the dynamic intracellular Ca(2+) concentration ([Ca(2+)](i)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca(2+)](i) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca(2+) stores, abolished these [Ca(2+)](i) responses in both osteoblasts and osteocytes, indicating that Ca(2+) release from intracellular stores plays a key role in the [Ca(2+)](i) oscillations of these bone cells in intact bone explants. Another possible [Ca(2+)](i) transient system to be considered is gap junctional communication through which Ca(2+) and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca(2+)](i) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca(2+)](i) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations that required the release of intracellular Ca(2+) stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca(2+)](i) oscillations of bone cells.     

Effects of neferine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits

To study the relaxation mechanisms of neferine (Nef) on the corpus cavernosum smooth muscle (CCSM), the CCSM cells from New Zealand White rabbits were cultured in vitro. [Ca(2+)](i) was measured by fluorescence ion digital imaging system (FIDIS), using Fluo-2/AM as a Ca(2+)-sensitive fluorescent indicator. Nef (0.1, 1 and 10 micromol l(-1)) had no effect on the resting [Ca(2+)](i) (P > 0.05). In the presence of extracellular Ca(2+) (2.5 mmol l(-1)), Nef (0.1, 1 and 10 micromol l(-1)) inhibited [Ca(2+)](i) elevation induced by high K(+) and phenylephrine (PE) in a concentration-dependent manner (P < 0.05). In calcium free solution containing egtaic acid (EGTA), Nef (0.1 micromol l(-1)) had no inhibitory effects on [Ca(2+)](i) elevation induced by PE (P > 0.05). However, Nef (1 and 10 micromol l(-1)) inhibited [Ca(2+)](i) elevation induced by PE (P < 0.05). These data suggest that Nef inhibited [Ca(2+)](i) in CCSM cells via blocking voltage-dependent Ca(2+) channel, alpha(1)-adrenoceptor-operated Ca(2+) channel and Ca(2+) release from intracellular Ca(2+) pool. This inhibitory action on [Ca(2+)](i) might be one of the relaxation mechanisms of Nef on the CCSM.     

Propofol inhibits Ca(2+) transients but not contraction in intact beating guinea pig hearts

We investigated whether propofol inhibits Ca(2+) transients and left ventricular pressure (LVP) in intact beating guinea pig hearts at clinical concentrations and whether an inhibition of Ca(2+) transients by propofol results from an impairment of sarcolemmal or of sarcoplasmic reticulum (SR) function. By using a Langendorff's preparation, transmural left ventricular phasic intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by the fluorescence ratio of indo-1 emission at 385 nm and 456 nm and was calibrated to Ca(2+) transients (in nM). The Ca(2+) transients during each contraction were defined as available [Ca(2+)](i). Sixty hearts were perfused with modified Krebs-Ringer's solution containing lipid vehicle and propofol (1 and 10 microM) in the absence and presence of ryanodine, thapsigargin, and nifedipine, while developed LVP and available [Ca(2+)](i) were recorded. Propofol (10 microM) decreased available [Ca(2+)](i) by 11.0% +/- 1.3% without decreasing developed LVP (% of control, P < 0.05). Propofol (10 microM) caused a leftward shift in the curve of developed LVP as a function of available [Ca(2+)](i). Propofol (10 microM) with nifedipine (1 microM), but not with ryanodine (1 microM) or thapsigargin (1 microM), decreased available [Ca(2+)](i) by 15.5% +/- 1.7% (P < 0.05). Propofol decreases available [Ca(2+)](i) without decreasing cardiac contraction, and it enhances myofilament Ca(2+) sensitivity in intact beating hearts at clinical concentrations. The inhibition of available [Ca(2+)](i) by propofol may be mainly mediated by an impairment of sarcoplasmic reticulum Ca(2+) handling rather than the sarcolemmal L-type Ca(2+) current. Implications: This is the first study of the effects of propofol on intracellular Ca(2+) concentration and myofilament Ca(2+) sensitivity under physiologic conditions in intact isolated beating guinea pig hearts.     

Prolonged calcium transients and myocardial remodelling in early experimental uraemia.

BACKGROUND: Cardiovascular disease is the most common cause of premature death in patients with end-stage renal disease, possibly due to a specific 'uraemic cardiomyopathy'. This study was designed to investigate the cardiac changes induced by a moderate impairment of renal function in a model of uraemia. METHODS: Male Wistar rats (n=11) were rendered uraemic by 5/6 nephrectomy or sham operated (n=11). After 4 weeks, cardiac dimensions were measured from fixed tissue sections using a digital image analysis technique. In parallel groups of animals, cardiac myocytes were isolated and studied for evidence of functional changes attributable to uraemia. After steady-state field stimulation at 0.5 Hz, intracellular Ca(2+) handling (using Fura-2) was investigated. Up to 20 consecutive transients were averaged as the extracellular Ca(2+) was increased. RESULTS: The 5/6 nephrectomy group had a 75% reduction in glomerular filtration rate, and a 2- to 3-fold increase in serum urea and creatinine compared with sham-operated control animals (P<0.0001). However, the blood pressure was found to be similar in each group. Histology of the intact hearts (five pairs) showed a significant increase in tissue cross-sectional area (14%; P<0.04), cross-sectional area of the left ventricle (22%; P<0.04), and a significant increase in left ventricular wall thickness (15%; P<0.03). In the single cardiac cell study, under basal conditions (1-2 mM extra-cellular Ca(2+)) no significant differences in intracellular Ca(2+) were observed, but in high extracellular Ca(2+) the uraemic cells were slower to return to diastolic intracellular Ca(2+) levels (P<0.05). CONCLUSIONS: The data provide evidence of altered myocardial structure and function in early experimental uraemia. The changes described are consistent with concentric hypertrophy of the left ventricle, which occurs in the absence of hypertension.     

Mechanism of the ropivacaine-induced increase in intracellular Ca2+ concentration in rat aortic smooth muscle

BACKGROUND: Ropivacaine is a long-acting local anesthetic with low cardiac toxicity that induces vasoconstriction in vitro and in vivo. Vascular smooth muscle tone is regulated by changes in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity. Therefore, the aim of this study was to examine the mechanism underlying the increase in [Ca(2+)](i) in ropivacaine-induced vascular contraction. METHODS: Ropivacaine-induced contractile responses and changes in [Ca(2+)](i) were examined using an isometric force transducer and a fluorometer, respectively. RESULTS: Ropivacaine induced a biphasic, concentration-dependent change in [Ca(2+)](i) and contractile response in rat aortic smooth muscles: an increase in [Ca(2+)](i) occurred at lower ropivacaine concentrations (3 x 10(-5) to 3 x 10(-4) M) and a decrease was observed at higher concentrations (10(-3) to 3 x 10(-3) M). Contraction and the [Ca(2+)](i) increase induced by ropivacaine were attenuated significantly by a voltage-dependent Ca(2+) channel antagonist, an inositol 1,4,5-triphosphate receptor antagonist and Ca(2+)-free solution (P < 0.01, n = 6). CONCLUSION: Ropivacaine-induced contraction of rat aortic smooth muscle is, in part, regulated by Ca(2+) influx from the extracellular space and Ca(2+) release from the sarcoplasmic reticulum.     

Morphine enhances myofilament CA(2+) sensitivity in intact guinea pig beating hearts

We investigated whether morphine alters intracellular Ca(2+) concentration ([Ca(2+)](i)), left ventricular pressure (LVP), and myofilament Ca(2+) sensitivity under physiologic conditions in intact guinea pig beating hearts and whether delta(1), delta(2), and kappa opioid stimulations are related to the direct cardiac effects of morphine. Transmural LV phasic [Ca(2+)](i) was measured from fluorescence signals at 385 nm and 456 nm. The Ca(2+) transients during each contraction were defined as available [Ca(2+)](i). The hearts were perfused with modified Krebs-Ringer solution containing morphine in the absence and presence of delta(1) (BNTX), delta(2) (NTB), and kappa (nor-BNI) antagonists, while developed LVP and available [Ca(2+)](i) were recorded. Morphine (1 microM) decreased available [Ca(2+)](i) by 44 +/- 12 nM without decreasing developed LVP at 2.5 mM of [CaCl(2)](e) (P < 0.05). Morphine (1 microM) caused a leftward shift in the curve of developed LVP as a function of available [Ca(2+)](i) (P < 0.05). BNTX (1 microM), but not nor-BNI (1 microM) or NTB (0.1 microM) blocked morphine (1 microM) effects to decrease available [Ca(2+)](i). Morphine decreases available [Ca(2+)](i) but not LVP, and it enhances myofilament Ca(2+) sensitivity under physiologic conditions at clinical concentrations in intact isolated beating guinea pig hearts. The delta(1) opioid stimulation modifies the effects of morphine on Ca(2+) transients and myofilament Ca(2+) sensitivity. IMPLICATIONS: Morphine modifies myofilament Ca(2+) sensitivity and Ca(2+) transients in guinea pig hearts at concentrations that are clinically relevant.     

Erythropoietin delivery by genetically engineered bone marrow stromal cells for correction of anemia in mice with chronic renal failure

The goal of this research was to develop a strategy to couple stem cell and gene therapy for in vivo delivery of erythropoietin (Epo) for treatment of anemia of ESRD. It was shown previously that autologous bone marrow stromal cells (MSCs) can be genetically engineered to secrete pharmacologic amounts of Epo in normal mice. Therefore, whether anemia in mice with mild to moderate chronic renal failure (CRF) can be improved with Epo gene-modified MSCs (Epo+MSCs) within a subcutaneous implant was examined. A cohort of C57BL/6 mice were rendered anemic by right kidney electrocoagulation and left nephrectomy. In these CRF mice, the hematocrit (Hct) dropped from a prenephrectomy baseline of approximately 55% to 40% after induction of renal failure. MSCs from C57BL/6 donor mice were genetically engineered to secrete murine Epo at a rate of 3 to 4 units of Epo/10(6) cells per 24 h, embedded in a collagen-based matrix, and implanted subcutaneously in anemic CRF mice. It was observed that Hct increased after administration of Epo+MSCs, according to cell dose. Implants of 3 million Epo+MSCs per mouse had no effect on Hct, whereas 10 million led to a supraphysiologic effect. The Hct of CRF mice that received 4.5 or 7.5 million Epo+MSCs rose to a peak 54+/-4.0 or 63+/-5.5%, respectively, at 3 wk after implantation and remained above 48 or 54% for >19 wk. Moreover, mice that had CRF and received Epo+MSCs showed significantly greater swimming exercise capacity. In conclusion, these results demonstrate that subcutaneous implantation of Epo-secreting genetically engineered MSCs can correct anemia that occurs in a murine model of CRF.     

Dynamic imaging of endoplasmic reticulum Ca2+ concentration in insulin-secreting MIN6 Cells using recombinant targeted cameleons: roles of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-2 and ryanodine receptors

The endoplasmic reticulum (ER) plays a pivotal role in the regulation of cytosolic Ca(2+) concentrations ([Ca(2+)](cyt)) and hence in insulin secretion from pancreatic beta-cells. However, the molecular mechanisms involved in both the uptake and release of Ca(2+) from the ER are only partially defined in these cells, and the presence and regulation of ER ryanodine receptors are a matter of particular controversy. To monitor Ca(2+) fluxes across the ER membrane in single live MIN6 beta-cells, we have imaged changes in the ER intralumenal free Ca(2+) concentration ([Ca(2+)](ER)) using ER-targeted cameleons. Resting [Ca(2+)](ER) (approximately 250 micromol/l) was markedly reduced after suppression (by approximately 40%) of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)-2b but not the SERCA3 isoform by microinjection of antisense oligonucleotides, implicating SERCA2b as the principle ER Ca(2+)-ATPase in this cell type. Nutrient secretagogues that elevated [Ca(2+)](cyt) also increased [Ca(2+)](ER), an effect most marked at the cell periphery, whereas inositol 1,4,5-trisphosphate-generating agents caused a marked and homogenous lowering of [Ca(2+)](ER). Demonstrating the likely presence of ryanodine receptors (RyRs), caffeine and 4-chloro-3-ethylphenol both caused an almost complete emptying of ER Ca(2+) and marked increases in [Ca(2+)](cyt). Furthermore, photolysis of caged cyclic ADP ribose increased [Ca(2+)](cyt), and this effect was largely abolished by emptying ER/Golgi stores with thapsigargin. Expression of RyR protein in living MIN6, INS-1, and primary mouse beta-cells was also confirmed by the specific binding of cell-permeate BODIPY TR-X ryanodine. RyR channels are likely to play an important part in the regulation of intracellular free Ca(2+) changes in the beta-cell and thus in the regulation of insulin secretion.     

Beneficial influence of recombinant human erythropoietin therapy on the rate of progression of chronic renal failure in predialysis patients.

BACKGROUND: Partial correction of anaemia with recombinant human erythropoietin (rHuEpo) has been shown to markedly improve the general condition and quality of life of predialysis patients, but the effects of rHuEpo therapy on blood pressure and the rate of progression of chronic renal failure (CRF) are still disputed. In particular, no study evaluated the time duration until the start of maintenance dialysis in treated patients, compared to untreated predialysis patients. METHODS: We retrospectively evaluated the rate of decline of creatinine clearance (Delta Ccr) and the duration of the predialysis period in 20 patients with advanced CRF treated with rHuEpo (Epo+ group), and in 43 patients with a similar degree of CRF but with less marked, asymptomatic anaemia, not requiring rHuEpo therapy (Epo- group). All patients were submitted to identical clinical and laboratory surveillance. All received similar oral supplementation with B(6), B(9), and B(12) vitamins and oral iron supplementation. Maintenance dose of subcutaneous epoetin was 54.3+/-16.5 U/kg/week (median dose 3300 U/week). RESULTS: Initial and final haemoglobin (Hb) levels were 8.8+/-0.7 and 11.3+/-0.9 g/dl in the Epo+ group, vs 10.9+/-1.2 and 9.5+/-0.9 g/dl in the Epo- group. In the Epo+ group, Delta Ccr declined from 0.36+/-0.16 during the preceding 24 months to 0.26+/-0.15 ml/min/ 1.73 m(2)/month after the start of rHuEpo therapy (P<0.05). No significant variation was observed in the Epo- group. Time duration until the start of dialysis was 16.2+/-11.9 in the Epo+ group, compared to 10.6+/-6.1 months in the Epo- group (P<0.01). Slowing of progression was observed in 10 Epo+ patients, whereas no significant variation in Delta Ccr occurred in the other 10. There was no difference in previous Delta Ccr rate, nor in Hb or blood pressure levels while on rHuEpo therapy between the two subgroups. CONCLUSIONS: Our study affords conclusive evidence that rHuEpo therapy did not result in accelerated progression of CRF in any treated predialysis patients, nor deleterious increase in blood pressure, but instead resulted in significant slowing of progression and substantial retardation of maintenance dialysis. Such encouraging results remain to be validated in a large prospective, randomized study.     

Effect of high glucose on basal intracellular calcium regulation in rat mesangial cell

BACKGROUND: A number of cellular mechanisms are critically dependent on intracellular Ca(2+) homeostasis. A sustained increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) is capable of activating a number of potentially harmful processes including phenotype change to secretory type, dysregulated cell proliferation, and cell injury and death. Mesangial cells (MCs) play an important role in the pathophysiology of diabetic nephropathy. METHODS: We evaluated the effect of high glucose on basal [Ca(2+)](i) in the unstimulated state and identified its contributing pathways. MCs were isolated and cultured from Sprague-Dawley rats. [Ca(2+)](i) was measured by fluorometric technique with fura-2AM. RESULTS: In a dose-dependent manner, superfusion of MCs with Tyrode's solution containing high glucose (30 and 50 mM) induced a delayed spontaneous increase in [Ca(2+)](i), which was not found in those with normal (5.5 mM) glucose or mannitol. The high glucose-induced increase in [Ca(2+)](i)()occurred through transmembrane influx of extracellular Ca(2+) and was blocked by SKF96365, an inhibitor of store-operated Ca(2+) influx. Na(+)-Ca(2+) exchanger (NCX) activity, a major channel regulating basal [Ca(2+)](i), and the clearing ability of intracellular Ca(2+) were depressed after MCs were cultured in high-glucose medium. Western blot analysis revealed the decreased expression of a 70-kD NCX protein in MCs cultured in high-glucose medium. CONCLUSIONS: A high-glucose concentration induced a spontaneous increase in basal [Ca(2+)](i) of MCs without stimulation. There was a decrease in the activity of NCX in the high-glucose condition, which seems to occur at the level of protein expression. The present results provide a novel insight into the mechanisms of diabetic nephropathy in that intracellular Ca(2+) homeostasis is an important secondary messenger and a mediator in hormonal signaling.