Low molecular weight iron in guinea pig reticulocytes

The low molecular weight iron found in the guinea pig reticulocyte has been partially characterized. On thin layer chromatography it is distinguishable from the iron complexes of a variety of nucleotides, sugars, and amino acids. On paper chromatography it comigrates with a 250-nm absorbing, orcinol-positive material. The eluted count peak contains phosphorus. Approximately 1 microgram of iron is recovered from 1 ml of hemolyzed red cells. Preparation under nitrogen improves recovery of low molecular weight iron, suggesting that the iron is in the ferrous oxidation state.     

Two pathways for iron uptake by guinea pig reticulocytes

We have demonstrated that the intracellular processing of transferrin to effect iron removal involves two pathways, one sensitive to rotenone and the other not. We have also found that the effect of the rotenone is dependent on the transferrin concentration: iron uptake was suppressed with concentrations of transferrin in the micromolar range, and was not suppressed at physiologic concentrations of transferrin. Rotenone does not disturb transferrin's interaction with its extracellular receptor, indicating that its action must be intracellular. The following model is suggested: that separate pathways are entered by transferrin in the cell. The first pathway is preferentially utilized when transferrin is in short supply. It begins with an intracellular site which has a high affinity (and low capacity) for either iron or transferrin. The second pathway begins with an intracellular site which has a high capacity (but low affinity) for either iron or transferrin and is utilized when transferrin is in physiologic concentration (and the low-capacity, high-affinity site is saturated); the pathway it initiates is dominant when transferrin is abundant. We speculate that the high-affinity low-capacity pathway may serve to direct intracellular iron to sites which would be critically injured by iron excess.     

Uptake of transferrin-bound and nontransferrin-bound iron by reticulocytes from the Belgrade laboratory rat: comparison with Wistar rat transferrin and reticulocytes.

The mechanism underlying the impaired uptake of iron from transferrin by reticulocytes from the Belgrade laboratory rat was investigated using 125I- and 59Fe-labeled transferrin isolated from homozygous Belgrade rats and from Wistar rats, nontransferrin-bound Fe(II) in an isotonic sucrose solution, and reticulocytes from Belgrade and Wistar rats. The Belgrade rat transferrin had the same molecular weight and net charge as Wistar rat transferrin, donated iron equally well to both types of reticulocytes, and competed equally for transferrin binding sites on the cells. Hence, the defect in iron uptake by Belgrade rat reticulocytes could not be attributed to an abnormality of the transferrin molecule. The rate of uptake of Fe(II) from sucrose into the cytosolic and stromal fractions of Belgrade rat reticulocytes was only about 35% as great as that by Wistar rat reticulocytes. With both types of cells, the uptake process was saturable, suggesting the presence of a carrier-mediated process. It was therefore concluded that the defect in iron uptake by Belgrade rat erythroid cells is probably the consequence of a deficiency in a membrane carrier for iron.     

Effects of ethanol on acid secretion by isolated parietal cells from guinea pig]

We studied the effects of ethanol (0.1-10%) on acid secretion of parietal cell-rich fractions isolated from guinea pig gastric mucosa. Ethanol (0.1-3%) increased histamine-stimulated cAMP content, while over 1% ethanol decreased histamine-stimulated acid secretion. H+, K(+)-ATPase activity in microsomal fraction also decreased after treatment with 3% ethanol. Thus, ethanol may disturb the signalling process from cAMP to H+, K(+)-ATPase. On the other hand, carbachol-stimulated acid secretion was more sensitive to ethanol than that with histamine, and 0.1% ethanol suppressed the acid secretion. This effect was well correlated with the extent of the ethanol-induced increase of [Ca2+]i and with the attenuation of [Ca2+]i response following carbachol stimulation. The calcium response may be a primary target against ethanol in carbachol-dependent process. In conclusion, low-dose ethanol have multi-effects on these critical intermediary steps in acid secretion.     

Iron proteins of duodenal enterocytes isolated from mice with genetically and experimentally altered iron metabolism

The molecular basis for the control of iron absorption by the duodenum remains unknown: however, ferritin (Ft) and the iron status of enterocytes have been suggested as regulatory factors. We determined the iron and Ft status of duodenal enterocytes from mice with hypotransferrinaemia, a genetic defect leading to greatly enhanced iron absorption, and for comparison we also investigated mice with experimentally-altered iron absorption. Duodenal enterocytes were isolated and analysed for Ft and non-haem iron content and for transferrin binding (as a measure of transferrin receptor activity). RNA was extracted from the duodenal mucosa and examined for transferrin receptor and H- and L-Ft mRNA levels by Northern hybridization analysis. Ft levels were elevated in enterocytes of hypotransferrinaemic mice, similar to that seen in iron dextran-injected mice of the CD1-strain. Enterocyte Ft levels were reduced in mice fed a diet diminished in iron, but unchanged in hypoxic mice enterocytes. Enterocytes of hypotransferrinaemic mice had normal non-haem iron levels and transferrin binding; however, enterocytes from CD-1 mice fed a low iron diet had increased transferrin binding and a decreased non-haem iron content. Duodenal mRNA levels for transferrin receptor and H-Ft were unchanged in hypotransferrinaemic mice, whereas L-Ft was increased. We conclude from the Ft and non-haem iron contents and transferrin binding that duodenal enterocytes from hypotransferrinaemic mice are not simply iron deficient, leading to increased expression of iron carriers proteins. Duodenal iron absorption can be enhanced in mice even when enterocyte Ft levels are raised or unchanged, suggesting that iron absorption is regulated by developmentally programmed expression of iron transporters by enterocytes.     

Mr 25,000 heparin-binding protein from guinea pig brain is a high molecular weight form of basic fibroblast growth factor.

A Mr 25,000 form of basic fibroblast growth factor (bFGF) has been isolated from guinea pig brain along with the typical Mr 18,000 form. Both forms were purified to homogeneity by a combination of heparin-affinity chromatography and ion-exchange chromatography on an FPLC Mono S column. The Mr 25,000 form, like the Mr 18,000 form, was not eluted from the heparin-affinity column with 0.95 M NaCl, but was eluted with 2 M NaCl. The Mr 25,000 guinea pig protein stimulated plasminogen activator production by cultured bovine capillary endothelial cells in a dose-dependent manner at concentrations of 0.1-10 ng/ml, the same range that was effective for guinea pig and human Mr 18,000 bFGFs. The binding of human 125I-labeled bFGF to baby hamster kidney cells is inhibited equally by the Mr 25,000 guinea pig protein and the Mr 18,000 guinea pig and human bFGFs. Polyclonal antibodies raised against human bFGF recognize both the Mr 25,000 and 18,000 guinea pig proteins in an immunoblot analysis. In a radioimmunoassay, both the Mr 25,000 and Mr 18,000 guinea pig proteins compete equally well with iodinated human bFGF for binding to the anti-human bFGF antibodies. When treated with low concentrations of trypsin, the Mr 25,000 guinea pig bFGF was converted to a Mr 18,000 protein. These results show that the two molecules are closely related and suggest that the Mr 25,000 protein shares substantial homology with the Mr 18,000 bFGF.     

Actions of genistein on contractile response of smooth muscle isolated from guinea pig gallbladder

BACKGROUND:Defective contractile motility of the gallbladder is an important factor for gallstone formation.Estrogen might increase the risk of gallstones and cholecystitis,and estradiol inhibits the contractile activity of isolated strips of guinea pig gallbladder.The potential risks associated with hormone replacement therapy(HRT) include symptomatic gallstones.Phytoestrogen have been used to treat menopause syndromes by replacing traditional estrogen.This experiment aimed to determine the effects of the ...     

Cortisol production by cells isolated from the outer and inner zones of the adrenal cortex of the guinea pig

Cortisol production by cells isolated from the outer (glomerulosa and fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig was evaluated in order to clarify the steroidogenic function of the zona reticularis. Basal cortisol production by the outer zone cells was 5-10 times greater than that by the inner zone cells. Cells from the outer zone increased cortisol production in response to ACTH (ED50, approximately 33 pg/ml), while cells from the inner zone failed to respond to ACTH. Likewise, cells from the outer zone increased cortisol production in response to (Bu)2cAMP, while cells from the inner zone failed to respond. When either pregnenolone or progesterone was added to the incubation mixture, an increase in cortisol production by cells of the inner zone was noted, although the increase was significantly less than that observed for cells from the outer zone. These results suggest that the zona reticularis of the guinea pig adrenal ordinarily produces little or no cortisol, although the enzyme machinery is present and capable of producing a limited amount of cortisol when supplied with the appropriate precursor.     

The induction of carbon monoxide-mediated airway relaxation by PACAP 38 in isolated guinea pig airways

Pituitary adenylate cyclase–activating peptide 38 (PACAP 38) displays several biologic activities relevant to obstructive airway disease. Carbon monoxide (CO) has recently emerged as a potent, endogenously produced mediator of bronchodilation. In this study, we have analyzed the occurrence of PACAP 38 and the corresponding occurrence of heme oxygenase (HO), the rate-limiting enzyme for CO production, in guinea pig trachea, using immunocytochemistry. We have also investigated whether the dilatory effects of PACAP 38 are dependent on CO, using an in vitro setup for tracheal studies. A moderate supply of PACAP-like immunoreactive nerve fibers was seen in association with tracheal smooth muscle. HO-like immunoreactivity was observed in the respiratory epithelium and in association with smooth muscle bundles. PACAP 38 induced a concentration-dependent relaxation of precontracted tracheal segments. This dilation was nearly abolished after pretreatment with zincprotoporphyrine, an inhibitor of heme oxygenase. The same effect was accomplished with Rp-8Br-cyclicGMPS, an inhibitor of cyclicGMP, whereas the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine had no effect on the PACAP 38–induced dilation. The presented data suggest that PACAP 38 can induce bronchodilation by means of a CO-dependent, cyclicGMP-related mechanism, thereby providing a link between neurotransmission and local CO release in the airway smooth muscle. Accepted for publication: 10 January 2001     

The calcium accumulating activity of subcellular fractions isolated from rat and guinea pig heart muscle.

Microsomal fractions prepared from rat ventricular muscle accumulated 23.15 ± 3.6 and 27.8 ± 2.4 nmol Ca²⁺/mg microsomal protein when incubated for 2 and 5 min respectively in an oxalate-free incubation medium, and 263.2 ± 26.0 and 327.3 ± 48.6 nmol Ca²⁺/mg microsomal protein when incubated for 2 and 5 min respectively in the presence of oxalate. These values for Ca²⁺ binding (absence of oxalate) and uptake (presence of oxalate) are significantly (P < 0.001) smaller than those obtained for guinea pig microsomal fractions.In the absence of Ca²⁺ the rat microsomal fractions hydrolysed ATP at a rate which was significantly faster than that exhibited by the guinea pig microsomes. Doses of 1 and 5 mm caffeine increased the peak tension developed by guinea pig and decreased the peak tension developed by rat papillary muscles. These same doses of caffeine prolonged the time required to develop peak tension in both species, and showed the rate of Ca²⁺ uptake. Doses of 1 and 5 mm caffeine slowed the rate at which the rat but not the guinea pig microsomes bound Ca²⁺ (in the absence of oxalate).These results are interpreted to mean that there is a species variation in the Ca²⁺-accumulating and ATPase activity of rat and guinea pig microsomal fractions.     

Enzyme release resulting from total ischemia and reperfusion in the isolated, perfused guinea pig heart.

A study of enzyme release induced by total myocardial ischemia was undertaken using isolated guinea pig hearts perfused at constant flow (4 ml/min). Mechanical activity (perfusion pressure and heart rate) and perfusate enzyme activity (malate dehydrogenase, lactate dehydrogenase and creatine phosphokinase) were examined throughout experiments in which the duration of total ischemia was varied (5, 10 and 20 min). The enzyme releases occurred generally in a biphasic pattern; a marked release just after reperfusion (first phase) and later a moderate release (second phase). The level of enzyme activity returned close to the control level within 7 min. The first phase of release was not accompanied by resumption of contraction. The rate of enzyme release correlated closely with the duration of total ischemia. The present results suggest that enzyme release during/after the tested durations of ischemia may not necessarily reflect serious myocardial cell damage, but rather alterations of the membrane permeability of myocardial cells.     

Assessment of vasoactive metabolites released from the isolated guinea pig during heart hypoxia and β-adrenergic stimulation

Summary Two isolated guinea pig hearts (H₁, H₂) were perfused in series in order to bioassay in the recipient heart (H₂) the release of vasoactive metabolites. Due to an effective reoxygenation system transit time between H₁ and H₂ as only 20 sec. Hypoxic perfusion (30% O₂) of H₁ caused relaxation of the coronary vessels of H₂, and this effect could be completely abolished by adenosine deaminase. Similar results were obtained when H₁ was stimulated by isoproterenol while H₂ was protected by propranolol. From our findings it is concluded that adenosine is the primary vasodilating substance released by the heart into the coronary circulation.     

Low molecular weight heparin-induced skin necrosis occurring distant from injection sites and without thrombocytopenia

Tietge UJF, Schmidt HH-J, Jäckel E, Trautwein C, Manns MP (Medizinische Hochschule Hannover, Hannover, Germany). Low molecular weight heparin-induced skin necrosis occurring distant from injection sites and without thrombocytopenia (Case Report). J Intern Med 1998; 243 : 313–15. In this paper we report a case of 76-year-old white male patient with skin necrosis induced by subcutaneous prophylactic administration of low-molecular-weight heparin (LMWH). Skin necrosis occured distant from heparin injection sites and without concomitant thrombocytopenia. This is the first reported case presenting these clinical findings.     

Effects ofl-propionylcarnitine on electrical and mechanical alterations induced by amphiphilic lipids in isolated guinea pig ventricular muscle

Summary We examined the effects ofl-propionyl-carnitine (Prop.C), a short-chain acylcarnitine, on amphiphile (l-lysophosphatidylcholine orl-palmitoylcarnitine)-induced electrophysiological and ultrastructural changes in isolated guinea pig ventricular papillary muscles, under acidic conditions (pH 6.9). Conventional microelectrode, tension-recording, and electron microscope techniques were used. Both amphiphiles, at a concentration of 10^–4 M, significantly decreased the resting membrane potential, action potential amplitude, and action potential duration, but increased the developed and resting tension. Such amphiphile-induced electrical changes were not observed in muscles pretreated with the beta-blocker, atenolol, although the mechanical changes remained unaffected. The application of Prop.C (10^–2 M), in the continued presence of the amphiphiles caused a return of the action potential duration and the developed tension to the control level. However, the resting potential and action potential amplitude remained unaffected; in fact, the maximum upstroke velocity ( ) of the action potential tended to decrease further. Pretreatment with Prop.C prevented all the amphiphile-induced electrophysiological and mechanical changes, except for . Electron microscopic studies revealed that amphiphile-induced ultrastructural changes were prevented, at least in part, in the presence of Prop.C. Thus, Prop.C antagonizes some of deleterious effects of amphiphiles, such as lysophosphatidylcholine and palmitoylcarnitine, upon the electrical and mechanical activities of the ventricular muscle, under acidic conditions.     

氢溴酸槟榔碱对豚鼠体外胃不同部位肌条作用及其机制

[目的]观察氢溴酸槟榔碱（Ah）对豚鼠体外胃不同部位平滑肌肌条收缩运动的影响，并初步探索其作用机制。[方法]用胃不同部位（6处）平滑肌条置于灌流浴槽，在37℃恒温、通氧和Krebs液灌注条件下，分别记录单独使用Ah和阻断剂孵育后Ah的收缩效应。[结果]①Ah可显著增高各部分体外胃平滑肌条的张力，增大收缩振幅和收缩波曲线下面积，2者均比基础值有显著增高（P〈0．05）。而且其效应强度不同部位有明显差异。纵肌效应：Ah对胃窦纵肌的作用振幅大于胃底、胃体（P〈0．01）；效应曲线下面积大于胃体（P〈0．01）。环肌效应：Ah对胃窦环肌作用振幅大于胃底（P〈0．05）；效应曲线下面积大于胃体和胃底（P〈0．05），对胃体环肌作用的曲线下面积亦大于胃底（P〈0．05）。②阿托品和维拉帕米均能部分阻断Ah的收缩作用，显著降低其振幅（P〈0．01）。[结论]Ah对豚鼠体外胃平滑肌条的收缩活动有明显兴奋作用，推测这种效应部分介导于M-胆碱能受体，L型电压依赖性Ca^2＋通道。     

Inhibition of glucose formation from fructose by phenformin in perfused guinea pig livers

Summary Glucose formation from fructose was inhibited by 4×10^–5 mol/1 phenylethylbiguanide (DBI) in perfused guinea pig livers. The pattern of hepatic metabolite concentrations revealed a cross-over phenomenon between fructose and fructose-1 -phosphate indicating that the phosphorylation of the substrate was influenced by the biguanide. It is suggested that this effect was due to a decrease of the ATP/ADP ratio observed in the presence of DBI.     

Dynamic modulation of Ca2+ sparks by mitochondrial oscillations in isolated guinea pig cardiomyocytes under oxidative stress

Local control of Ca²⁺-induced Ca²⁺ release (CICR) depends on the spatial organization of L-type Ca²⁺ channels and ryanodine receptors (RyR) in the dyad. Analogously, Ca²⁺ uptake by mitochondria is facilitated by their close proximity to the Ca²⁺ release sites, a process required for stimulating oxidative phosphorylation during changes in work. Mitochondrial feedback on CICR is less well understood. Since mitochondria are a primary source of reactive oxygen species (ROS), they could potentially influence the cytosolic redox state, in turn altering RyR open probability. We have shown that self-sustained oscillations in mitochondrial inner membrane potential (ΔΨ_m), NADH, ROS, and reduced glutathione (GSH) can be triggered by a laser flash in cardiomyocytes. Here, we employ this method to directly examine how acute changes in energy state dynamically influence resting Ca²⁺ spark occurrence and properties. Two-photon laser scanning microscopy was used to monitor cytosolic Ca²⁺ (or ROS), ΔΨ_m, and NADH (or GSH) simultaneously in isolated guinea pig cardiomyocytes. Resting Ca²⁺ spark frequency increased with each ΔΨ_m depolarization and decreased with ΔΨ_m repolarization without affecting Ca²⁺ spark amplitude or time-to-peak. Stabilization of mitochondrial energetics by pretreatment with the superoxide scavenger TMPyP, or by acute addition of 4′-chlorodiazepam, a mitochondrial benzodiazepine receptor antagonist that blocks the inner membrane anion channel, prevented or reversed, respectively, the increased spark frequency. Cyclosporine A did not block the ΔΨ_m oscillations or prevent Ca²⁺ spark modulation by ΔΨ_m. The results support the hypothesis that mitochondria exert an influential role on the redox environment of the Ca²⁺ handling subsystem, with mechanistic implications for the pathophysiology of cardiac disease.     

Bacteria capture iron from heme by keeping tetrapyrrol skeleton intact

Because heme is a major iron-containing molecule in vertebrates, the ability to use heme-bound iron is a determining factor in successful infection by bacterial pathogens. Until today, all known enzymes performing iron extraction from heme did so through the rupture of the tetrapyrrol skeleton. Here, we identified 2 Escherichia coli paralogs, YfeX and EfeB, without any previously known physiological functions. YfeX and EfeB promote iron extraction from heme preserving the tetrapyrrol ring intact. This novel enzymatic reaction corresponds to the deferrochelation of the heme. YfeX and EfeB are the sole proteins able to provide iron from exogenous heme sources to E. coli. YfeX is located in the cytoplasm. EfeB is periplasmic and enables iron extraction from heme in the periplasm and iron uptake in the absence of any heme permease. YfeX and EfeB are widespread and highly conserved in bacteria. We propose that their physiological function is to retrieve iron from heme.     

采用分子筛层析法分离纯化细粒棘球蚴囊液粗抗原

目的分离纯化细粒棘球蚴囊液抗原,去除其中的非特异性反应蛋白,探索优化囊液抗原制备方法。方法采用超滤法浓缩羊肝细粒棘球蚴囊液制备成粗抗原,并采用免疫印迹实验对囊液抗原进行分析,发现非特异性反应的主要蛋白条带。随后采用Superdex凝胶柱通过分子筛层析法对囊液抗原进行纯化。用44份细粒棘球蚴病患者、17份健康人和10份囊尾蚴患者血清进行酶联免疫实验,评估纯化后囊液抗原的检测能力。结果免疫印迹实验发现,非特异性反应的主要条带的相对分子质量(M_r)约为55 000,纯化后去除了该非特异性反应蛋白。纯化后的囊液抗原ELISA检测灵敏度为93.2%(41/44),特异性为94.1%(16/17),约登指数为0.87,与囊尾蚴病交叉反应性为30%(3/10)。未纯化前的灵敏度为90.9%(40/44),特异性为88.2%(15/17),约登指数0.79,与囊尾蚴病交叉反应性为60%(6/10)。纯化前后ELISA检测的灵敏度存在显著性差异。结论本研究采用分子筛层析法对羊源细粒棘球蚴的囊液抗原进行纯化,去除了引起非特异性反应的主要蛋白(M_r 55 000),为后续进行囊液抗原标准化、提高检测能力提供了依据。     

Leukotrienes stimulate pepsinogen secretion from guinea pig gastric chief cells by a nitric oxide-dependent pathway

Leukotrienes (LTs) are involved in many inflammatory conditions including gastric damage induced by nonsteroidal anti-inflammatory drugs. Although LTs stimulate acid secretion, the effect they exert on pepsinogen secretion is unknown. The aim of this study was to investigate whether LTs stimulate pepsinogen secretion by isolated chief cells and to identify the intracellular messengers that mediate this action. Isolated chief cells were incubated with concentrations of LTB₄, LTC₄, LTD₄, or LTE₄ ranging from 0.1 pmol/L to 10 μmol/L, and pepsinogen release, intracellular calcium and inositol(1,4,5)-trisphosphate (IP₃) concentrations were measured. Nitric oxide generation was determined by the amount of citrulline generated during incubation. All four LTs caused a concentration-dependent stimulation of pepsinogen secretion with 50% effective concentration of 0.05-0.1 nmol/L and a dose-dependent increase in cytoplasmic free calcium and IP₃ concentration. The LTB₄ and LTD₄ antagonists caused selective, concentration-dependent inhibition of LTB₄- and LTD₄-induced pepsinogen secretion, calcium mobilization, and IP₃ generation. All four LTs increased NO generation, and the effect was inhibited by LTB₄ and LTD₄ antagonists and an NO synthase inhibitor N^G-monomethyl-l-arginine and reversed by l-arginine. N^G-monomethyl-l-arginine caused a 50%–60% reduction of LT-induced pepsinogen release. Each of the four LTs caused a fivefold increase in 5′-cyclic guanosine monophosphate. LTs are powerful stimulators of pepsinogen secretion in isolated chief cells and act via occupancy of specific cell-surface receptors.