Ca Outflow Research Articles

Duration of forest fertilization effects on streamwater chemistry in a catchment in central Sweden

Demands for forest biomass production for energy, construction and carbon storage purposes are increasing, and therefore measures to increase tree growth are required. One potential measure is nitrogen (N) fertilization, as N is usually the most growth-limiting nutrient in boreal forests and partly due to decreasing atmospheric N deposition in northern Europe in recent decades. However, N fertilization can have adverse effects, such as soil acidification and N leaching, particularly nitrate leaching via streamwater flow. To mitigate the acidification risk, dolomite (CaMg(CO3)2) is added to N fertilizer boron (B) as increased tree growth hamper tree uptake of this essential micronutrient.This study examined the effects of forest fertilization on streamwater chemistry in the 45 ha Swedish catchment Risfallet (RF), around 80% of which was treated with fertilizer. That was rather exceptional, as most previous catchments studied have had <50% treated area, which may give weaker treatment signals. A paired catchment method combined with the control area and calibration period technique was applied to evaluate leaching effects from forest fertilization.Effects over 7.5 years were compared with previously reported initial effects in the first year, in order to assess the duration of fertilization effects on surface water. High excess outflow of N over five years was detected, with 20% of the applied amount leached and with nitrate dominating total nitrogen. Excess outflow of Ca and Mg was highest in the first year. Effects on pH were limited, with calculated untreated pH on 5.9 being on average 0.4 units lower during the first six months and then remaining at 0.2 units lower.Recommended could be to mainly fertilize well-drained soil, avoid wet areas and open streams. Consider the hydrological conditions while weather would be more hazardous to foresee.

Efficiency of 3D Implants with Bioactive Properties for Treatment of Extensive Bone Defects: Experimental Study

Background. The problem of replacing extensive bone defects remains relevant. The use of implant structures with bioactive properties can stimulate osteogenesis, which will improve the final treatment result.The aim of the study. In an in vivo experiment, to study the possibility of replacing an extensive defect in the bone diaphysis with a personal bioactive cellular 3D implant and evaluate the long-term results of its use.Materials and Methods. In an in vivo experiment, adult large mongrel dogs (n = 8) were modeled with an extensive segmental defect of the tibial diaphysis measuring 4 cm. The defect was replaced with a cellular bioactive 3D implant made of titanium alloy Ti6Al4V, manufactured using the additive technology. The diameter of the cells was 1.5 mm on average. The walls of the implant had pores of 100– 300 μm in size. The inner and outer surfaces were coated with a calcium phosphate layer formed by micro-arc oxidation. The primary fixation was provided with the Ilizarov apparatus. In the early postoperative period, antibiotic prophylaxis with broad-spectrum drugs was performed. Clinical, X-ray, histological and statistical methods were used to analyze the results. The main control points were considered: the end of external fixation with the Ilizarov apparatus, after 180 days and 1 year after the termination of external fixation.Results. During the experiment, the death of animals and complications were not observed. The spatial location of the implant was preserved. The formation of a strong bone-implantation block occurred 37.2±6.3 days after the operation. During this period, the external fixation apparatus was dismantled. Osseointegration was provided under conditions of sufficient primary mechanical stability, due to the cellular structure of the implant, the presence of pores on its walls, and the osteoinductive properties of the applied calcium phosphate coating. The achieved degree of osseointegration persisted in long-term periods (6 months and 1 year after the termination of external fixation). The osteoinductive properties of the calcium phosphate coating were confirmed by the expression of osteopontin cells at all stages of the experiment. Outflow of Ca and P from bone fragments was not observed. An elastic sheath was formed on the surface of the implant, similar in structure to the periosteum. The implant cells were filled with a well-vascularized bone substrate. In the projection of the intermediate zone, compact bone tissue was formed, and in the projection of the medullary canal — reticulofibrous bone marrow. This indicates the possibility of organotypic remodeling of bone structures inside the implant.Conclusion. The results of the study showed the effectiveness of using a bioactive cellular 3D implant to replace an extensive defect in the shaft of the bone. The architectonics and osteoinductive properties of the implant surface contributed to the formation of complete osseointegration in a short time, while maintaining the achieved result in long-term periods.

Adrenal Medulla Chemo Sensitivity Does Not Compensate the Lack of Hypoxia Driven Carotid Body Chemo Reflex in Guinea Pigs.

Guinea pigs (GP), originally from the Andes, have absence of hypoxia-driven carotid body (CB) reflex. Neonatal mammals have an immature CB chemo reflex and respond to hypoxia with metabolic changes arising from direct effects of hypoxia on adrenal medulla (AM). Our working hypothesis is that adult GP would mimic neonatal mammals. Plasma epinephrine (E) has an AM origin, while norepinephrine (NE) is mainly originated in sympathetic endings, implying that specific GP changes in plasma E/NE ratio, and in blood glucose and lactate levels during hypoxia would be observed. Experiments were performed on young adult GP and rats. Hypoxic ventilation (10% O2) increased E and NE plasma levels similarly in both species but PaO2 was lower in GP than in rats. Plasma E/NE ratio in GP was higher (≈1.0) than in rats (≈0.5). The hypoxia-evoked increases in blood glucose and lactate were smaller in GP than in the rat. The AM of both species contain comparable E content, but NE was four times lower in GP than in rats. GP superior cervical ganglion also had lower NE content than rats and an unusual high level of dopamine, a negative modulator of sympathetic transmission. Isolated AM from GP released half of E and one tenth of NE than the rat AM, and hypoxia did not alter the time course of CA outflow. These data indicate the absence of direct effects of hypoxia on AM in the GP, and a lower noradrenergic tone in this species. Pathways for hypoxic sympatho-adrenal system activation in GP are discussed.

Preparation of antibacterial chlorhexidine/vermiculite and release study

The antibacterial chlorhexidine/vermiculite (CA/Ver) was successfully prepared through the intercalation process and the stability of CA on the vermiculite matrix and was investigated by stirring in aqueous solutions under the influence of different pH and temperature. The content of CA was determined by total organic carbon (TOC) analysis before and after stability tests. The structure of all samples was characterized by X-ray powder diffraction (XRD) and Fourier–transform infrared spectroscopy (FTIR). The antibacterial activity of prepared CA/Ver samples was evaluated by finding a minimum inhibitory concentration (MIC) against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The content of chlorhexidine ranged from 209 to 231.6mg of CA in 1g of the whole sample after the intercalation process. After stability study, only a slight outflow of CA from the Ver matrix (<5%) was noted. The antibacterial test confirmed that the outflow of CA was negligible.After 30min of exposition the MIC of organovermiculite samples before and after stability test were the same for Staphylococcus aureus and Escherichia coli with value 3.33 (%; w/v) and the MIC decreased to 0.014 (%; w/v) with longer time of exposition (120h). A small difference was observed at Enterococcus faecalis where MIC was 10 (%; w/v) after 30min of exposition for the sample after stability test in neutral pH. However, after 24h of treatment the MIC value decreased to 0.014 (%; w/v). And finally, bacterial strain Pseudomonas aeruginosa showed a great resistance against antibacterial organovermiculite samples and MIC did not decreased under 10 (%; w/v) even after 5days of exposition.

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K + (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K + in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86 Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45 Ca outflow provoked by a rise in the extracellular K + concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K + challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca 2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca 2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca 2+ channels.

Tricyclic antidepressant imipramine reduces the insulin secretory rate in islet cells of Wistar albino rats through a calcium antagonistic action.

Treatments with antidepressants have been associated with modifications in glucose homeostasis. The aim of this study was to assess the effect of imipramine, a tricyclic antidepressant, on insulin-secreting cells. Insulin radioimmunoassay, radioisotopic, fluorimetric and patch-clamp methods were used to characterise the effects of imipramine on ionic and secretory events in pancreatic islet cells from Wistar albino rats. Imipramine induced a dose-dependent decrease in glucose-stimulated insulin output (IC(50)=5.2 micromol/l). It also provoked a concentration-dependent reduction in (45)Ca outflow from islets perifused in the presence of 16.7 mmol/l glucose. Moreover, imipramine inhibited the increase in (45)Ca outflow mediated by K(+) depolarisation. Patch-clamp recordings further revealed that imipramine provoked a marked and reversible decrease of the inward Ca(2+) current. In single islet cells, imipramine counteracted the rise in [Ca(2+)](i) evoked by glucose or high K(+) concentrations. These data indicate that imipramine dose-dependently reduces the insulin secretory rate from rat pancreatic beta cells. Such an effect appears to be mediated by the inhibition of voltage-sensitive Ca(2+) channels with subsequent reduction in Ca(2+) entry. Thus, it is possible that some adverse effects of imipramine are related, at least in part, to its capacity to behave as a Ca(2+) entry blocker.

Maize tonoplast PP i-dependent H +/Ca 2+ exchange: two Ks for Ca 2+ and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca 2+ using the energy derived from the H + gradient formed during PP i hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca 2+ accumulated by tonoplast. Two apparently different K s values for Ca 2+ with values of 0.36 and 4.70 μM were detected when oxalate was included in the medium and the free Ca 2+ concentration in the medium was buffered with the use of EGTA. Binding of Ca 2+ to the outer surface of tonoplasts inhibits the outflow of Ca 2+ previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 μM Ca 2+. Thapsigargin, a specific inhibitor of Ca 2+-ATPase, inhibits the Ca 2+ uptake driven by H + gradient but does not inhibit the hydrolysis of PP i nor the formation of a H + gradient.

In vitro and in vivo effects of new insulin releasing agents

The present study aimed at characterizing in vitro and in vivo the effects of BM 208 ( N-[4-(5-chloro-2-methoxybenzamidoethyl)benzenesulfonyl]- N′-cyano- N″-cyclohexylguanidine) and BM 225 (1-[4-(5-chloro-2-methoxybenzamidoethyl)benzene sulfonamido]-1-cyclohexylamino-2-nitroethylene); two new isosteres of the hypoglycemic sulfonylurea glibenclamide. In rat pancreatic islets perifused at close to normal (8.3 mM) d-glucose concentration, both BM 208 and BM 225 (10 and 25 μM) increased 45 Ca outflow and insulin release. The compounds did not affect the 45 Ca outflow rate from islets exposed to Ca 2+-free media. In single pancreatic islet cells loaded with the fluorescent Ca 2+ indicator fura-2 and incubated in the presence of 8.3 mM glucose, BM 208 and BM 225 raised the [Ca 2+] i. All these findings indicate that, in islet cells exposed to a physiological concentration of d-glucose, the secretory capacity of the new glibenclamide isosteres is related to a facilitation of Ca 2+ entry. The potency and duration of action of BM 225 was, however, more pronounced than that of BM 208. Successive additions of BM 208 provoked repeated increments in 45 Ca outflow and insulin release, without evidence of tachyphylaxis. Lastly, intraperitoneal injection of BM 208 and BM 225 to fed rats lowered plasma glucose concentration in a dose-dependent manner. BM 225 was more potent and acting faster than BM 208. Our results indicate that appropriate structural modification can generate isosteres of glibenclamide with different features and activity profiles.

Synthesis and characterization of a quinolinonic compound activating ATP-sensitive K(+) channels in endocrine and smooth muscle tissues.

Original quinolinone derivatives structurally related to diazoxide were synthesized and their effects on insulin secretion from rat pancreatic islets and the contractile activity of rat aortic rings determined. A concentration-dependent decrease of insulin release was induced by 6-chloro-2-methylquinolin-4(1H)-one (HEI 713). The average IC(50) values were 16.9+/-0.8 microM for HEI 713 and 18.4+/-2.2 microM for diazoxide. HEI 713 increased the rate of (86)Rb outflow from perifused pancreatic islets. This effect persisted in the absence of external Ca(2+) but was inhibited by glibenclamide, a K(ATP) channel blocker. Inside-out patch-clamp experiments revealed that HEI 713 increased K(ATP) channel openings. HEI 713 decreased (45)Ca outflow, insulin output and cytosolic free Ca(2+) concentration in pancreatic islets and islet cells incubated in the presence of 16.7 or 20 mM glucose and extracellular Ca(2+). The drug did not affect the K(+)(50 mM)-induced increase in (45)Ca outflow. In aortic rings, the vasorelaxant effects of HEI 713, less potent than diazoxide, were sensitive to glibenclamide and to the extracellular K(+) concentration. The drug elicited a glibenclamide-sensitive increase in (86)Rb outflow from perifused rat aortic rings. Our data describe an original compound which inhibits insulin release with a similar potency to diazoxide but which has fewer vasorelaxant effects. Our results suggest that, in both aortic rings and islet tissue, the biological effects of HEI 713 mainly result from activation of K(ATP) channels ultimately leading to a decrease in Ca(2+) inflow.

Effect of the absence of bicarbonate upon intracellular pH and calcium fluxes in pancreatic islet cells

The removal of extracellular HCO − 3 together with a decrease in pCO 2, in order to maintain a normal extracellular pH, caused a sustained increase of intracellular pH in rat pancreatic islets. This increase was more marked in glucose-deprived than in glucose-stimulated islets, and was associated with a facilitation of 45Ca efflux from the glucose-deprived islets. Such a facilitation was slightly reduced in the absence of extracellular Ca 2+ and abolished at low extracellular Na + concentration. It failed to occur in glucose-stimulated islets, whether in the presence or absence of extracellular Ca 2+. The removal of HCO − 3 and decrease in the pCO 2 also reduced the magnitude of both the secondary rise in 45Ca efflux and stimulation of insulin release normally evoked by an increase in glucose concentration. These findings suggest that changes in intracellular pH affect both the outflow of Ca 2+ from islet cells as mediated by Na +-Ca 2+ countertransport and the inflow of Ca 2+ by gated Ca 2+ channels. The experimental data are also compatible with the view that islet cells are equipped with an active process of bicarbonate-chloride exchange involved in the regulation of intracellular pH.

A kinetic analysis of the effects of adrenaline on calcium distribution in isolated rat liver parenchymal cells.

1. The effects of adrenaline on Ca distribution in isolated rat liver parenchymal cells were studied using a (45)Ca exchange technique under steady-state conditions with respect to the net movement of Ca. (45)Ca was initially introduced into the extracellular medium. The amount of cellular (45)Ca was determined after separation of the cells from the medium by centrifugation through a solution which contained LaCl(3) (to displace (45)Ca bound to sites on the outside of the cell membrane) and silicon oil. At 1.3 and 2.4 mm-extracellular Ca, a stimulation of the initial rate of (45)Ca exchange was observed in the presence of 10(-7)m-adrenaline (or 10(-6)m-phenylephrine) with a 7% decrease, and no change, respectively, in the plateau of the exchange curve. The same degree of stimulation was observed when (45)Ca was added at 1, 15, 30 or 45 min after the adrenaline.2. No stimulation of the initial rate of exchange was observed at 0.1 mm-extracellular Ca, or at 2.4 mm-extracellular Ca in the presence of antimycin A and oligomycin. At 0.1 mm-Ca, a 60% decrease in the plateau of the exchange curve was observed in the presence of adrenaline. The concentration of adrenaline (10(-7)m) which caused half-maximal stimulation of the initial rate of (45)Ca exchange at 1.3 mm-Ca was similar to that (2 x 10(-7)m) which caused half-maximal decrease in the plateau at 0.1 mm-Ca.3. The addition of adrenaline to cells equilibrated with (45)Ca at either 2.4 or 1.3 mm-Ca caused a transient loss of (45)Ca followed by a return to a new steady state after 1 or 10 min, respectively. A loss of (45)Ca was also observed at 0.1 mm-Ca, but the (45)Ca content of the cells remained maximally depressed for at least 30 min.4. A non-linear least-squares iterative curve-fitting technique was used to demonstrate that (a) an equation which includes two exponential terms and (b) a parallel or series arrangement of three compartments of exchangeable Ca (the medium and two compartments associated with the cell) are consistent with each set of data obtained at 1.3 or 2.4 mm-Ca in the presence or absence of adrenaline (or phenylephrine). At 1.3 mm-Ca, the quantities of exchangeable Ca in the two kinetically defined cellular compartments were 0.04-0.07 and 0.34-0.37 nmol per mg wet weight with rate constants for Ca outflow of 1.2-1.5 and 0.06-0.08 min(-1), respectively.5. Analysis of the changes induced by adrenaline or phenylephrine showed that at 1.3 and 2.4 mm-extracellular Ca these agents caused a 75-150% increase in the quantity of exchangeable Ca in the small kinetically defined compartment and a 20% decrease in the quantity of exchangeable Ca in the large kinetically defined compartment. These changes were mediated by an 80-160% increase in the rate constant for the inflow of Ca from the medium to the small kinetically defined compartment, and either a 20-60% decrease in the rate constant for inflow to, or a 20% increase in the rate constant for outflow from, the large compartment.6. Replacement of the LaCl(3) in the solution used to separate the cells from the incubation medium with either 5 mm-EGTA or 5 mm-CaCl(2) did not alter the kinetics of (45)Ca exchange or the stimulation by adrenaline. This, together with the observation that at 1.3 mm-extracellular Ca, adrenaline increases the initial rate of exchange in the absence, but not in the presence, of antimycin A plus oligomycin, indicates that both cellular compartments of exchangeable Ca are intracellular.7. The addition of antimycin A plus oligomycin to cells equilibrated with (45)Ca at 2.4 mm-extracellular Ca in the presence or absence of adrenaline displaced 0.09 and 0.14 nmol (45)Ca. mg(-1), respectively.8. Subcellular fractionation of cells equilibrated with (45)Ca at 0.1 mm-extracellular Ca revealed that the mitochondria and microsomes contained significant amounts of (45)Ca. The amounts of (45)Ca in these fractions decreased by 50 and 40%, respectively, in the presence of adrenaline.9. In (45)Ca exchange experiments conducted with isolated mitochondria at 37 degrees C at 1.5 x 10(-7)m and 0.9 x 10(-7)m free Ca in the presence of 2 mm-Mg(2+), one kinetically defined compartment of exchangeable mitochondrial Ca was detected. The rate constants for Ca outflow were found to be 0.15+/-0.03 and 0.12+/-0.04 min(-1), respectively, in reasonable agreement with the value obtained for the rate constant for the outflow of Ca from the large kinetically defined compartment of exchangeable Ca observed in cells.10. It is concluded that adrenaline has two effects on Ca movement in the liver cell. These are to cause a loss of Ca from an intracellular compartment, which includes the mitochondria and microsomes, and to increase the transport of Ca from the extracellular medium to an intracellular site. This results in an increase in the amount of Ca in a small intracellular compartment which may represent cytoplasmic Ca, or Ca bound to sites on the inside of the plasma membrane.

Calcium transport and ATPase activity in mitochondria and fragments of sarcoplasmic reticulum of the heart and muscles of rabbits with hypercholesteremia

Keeping rabbits on a high-cholesterol diet (1 g/kg) for 3–7 months led to an increase in cholesterol concentration in the mitochondrial membranes and fragments of the sarcoplasmic reticulum (SPR) of the myocardium and skeletal muscles. Saturation of the membranes with cholesterol led to a decrease in efficiency of the Ca-pump of the SPR, as reflected in lowering of the Ca/ATP ratio and an increase in the outflow of Ca++ from the SPR. Under these conditions the rate of accumulation of Ca++ was higher in SPR than in the mitochondria. Activity of mitochondrial Mg++-activated 2,4-DNP-ATPase was reduced in hypercholesteremia.

Some properties of the calcium pump of the sarcoplasmic reticulum of the skeletal muscles of rabbits with hypercholesteremia

Fragments of sarcoplasmic reticulum (SR) isolated from the skeletal muscles of rabbits with marked atherosclerosis have impaired ability to accumulate Ca++. A decrease in the efficiency of the transport process, expressed as a decrease in the Ca/ATP ratio, is accompanied by activation of Ca-ATPase and by a simultaneous increase in the rate of outflow of Ca++ from SR. The temperature dependence of Ca-ATPase, plotted between Arrhenius coordinates, normally has an inflection at 20–21°C, but in hypercholesteremia the graph becomes a straight line. Meanwhile the cholesterol/protein ratio in membrane preparations of SR rises sharply in atherosclerosis. The Ca-ATPase of native membranes has an activation energy (Ea) of 15.6 and 28.7 kcal/mole in regions above and below the inflection respectively. The Ca-ATPase of membranes containing an increased amount of cholesterol has an activation energy of 19 kcal/mole over the whole range of temperatures investigated. It is suggested that the cholesterol level in membrane preparations changes not only the physicochemical characteristics of the membranes, but also the enzymic properties of transport ATPase.

Investigation of the nature of differences between ?-adrenergic receptors

The results of an investigation of the affinity of propranolol, alprenolol, and practolol for β-adrenergic receptors of various organs confirm the division of these receptors into two subtypes. The action of isoprenaline on β1-adrenergic receptors of the smooth muscles of the gastrointestinal tract and myocardium is accompanied by a change in the inflow of Ca++ ions into the cells. Under the influence of isoprenaline on β2-adrenergic receptors of the vessels and trachea an increase in the outflow of Ca++ ions from the cells and a decrease in tone of the smooth muscles are observed.

ATP-independent calcium net movements in human red cell ghosts.

Net Ca movements in metabolically depleted red cell ghosts were measured under the influence of different inwardly and outwardly directed Ca concentration gradients. Four variants of potassium-rich ghosts were prepared by reversal of osmotic hemolysis: (1) OCa-ghosts (hemolyzing medium (HM) contained no Ca); (2) OCa-EGTA-ghosts (HM contained 2 to 4mm EGTA1 and no Ca); (3) Ca-ghosts (HM contained 0.1 to 4.0mm Ca); and (4) Ca-EGTA-ghosts [HM contained Ca and EGTA in various proportions ([Ca]<[EGTA])]. Ca uptake in OCa- and OCa-EGTA-ghosts at any particular extracellular concentration ([Ca]0) within 60 min reached a steady state when cellular Ca and [Ca]0 were still far from equilibration. About half of the total Ca taken up by OCa-EGTA-ghosts penetrated into the cell interior whereas the other half seemed to be bound to membrane sites. The amount of cellular Ca uptake (i.e., uptake into the cell interior as well as binding to the membrane) decreased with increasing internal concentration of ionized Ca. With 10−6m internal free Ca, the uptake was nearly completely inhibited. In OCa-EGTA-ghosts the amount of Ca taken up into the intracellular space was markedly increased by the addition of mersalyl (0.05mm) to the external medium. Ca outflow under outward gradients was larger than inflow under inward concentration gradients of similar magnitude and no steady state was reached within 180 min. The incorporation of more than 5mm Ca into red cell ghosts led to a general increase in membrane permeability and finally to hemolysis of the ghosts. Lanthanum was found to inhibit Ca uptake in OCa-and OCa-EGTA-ghosts. The experimental results suggest that membrane-bound Ca regulates the Ca permeability of the red cell membrane. In the absence of ATP the uptake of Ca into red cell ghosts in contrast to outflow is a self-limiting process. This mechanism, in addition to active outward Ca pumping, may help to keep the free internal Ca in red cells at a low level.