Calcium-free Perfusion Research Articles

Extracellular levels of ATP and acetylcholine during lithium-pilocarpine induced status epilepticus in rats

Acetylcholine (ACh) and ATP are rapidly acting neurotransmitters with a putative role in epileptic seizures. In the present study we investigated extracellular concentrations of both neurotransmitters in parallel by microdialysis in rat hippocampus. We found that infusion of neostigmine increases, while calcium-free perfusion and infusion of tetrodotoxin (TTX) decreases, ACh levels. Calcium-free perfusion also decreased ATP levels which were, however, not affected by neostigmine or TTX. During status epilepticus, ACh levels were increased threefold but returned to baseline after the termination of seizures by diazepam. ATP levels were unchanged during status epilepticus but a several-fold increase was seen when AOPCP, an inhibitor of 5′-endonucleotidase, was infused. The results demonstrate an increase of ATP levels during epileptic seizures which, however, was not of neuronal origin.

Pentobarbital Decreased Nitric Oxide Release in the Rat Striatum but Ketamine Increased the Release Independent of Cholinergic Regulation

Pentobarbital (PB) and ketamine (Ket) influence the concentration of neurotransmitters in the brain. PB has been reported to decrease the extracellular nitric oxide (NO) concentration through a decrease in acetylcholine (ACh) release, while Ket has been shown to increase the NO concentration via an increase in ACh release. Here, we investigated effects of PB and Ket on NO release and the relationship between NO and ACh in the rat striatum by in vivo microdialysis experiments. Male Sprague-Dawley rats were used. A microdialysis probe was inserted into the right striatum and perfused with modified Ringer's solution. Samples were collected every 15 min and injected into an HPLC system. The rats were freely moving, and PB and Ket were administered intraperitoneally. Neostigmine (1 and 10 µM) and mecamylamine (100 µM) were added to the perfusate. Calcium and magnesium concentrations were modified for each anesthetic to influence ACh release. PB decreased NO products (NOx) while Ket increased them. While perfusion with neostigmine showed no effect on baseline NOx concentrations, it diminished the PB-induced NOx reduction at low concentrations and abolished it at high concentrations. Magnesium-free perfusion had no effect on baseline NOx concentrations, whereas perfusion at a low magnesium concentration antagonized the PB-induced NOx reduction. Mecamylamine and calcium-free perfusion had no effect on baseline NOx concentrations and Ket-induced NOx increases. PB may decrease NO release through reduction in ACh release, whereas Ket may increase NO release independent of ACh regulation.

Study on vasodilatation effects of total flavonoid from polygonum aviculare on blood vessel of rat in vitro

Objective To investigate the vasodilatation effects of total flavonoid from polygonum aviculare on rat thoracic aorta and its underlying mechanisms. Methods Total flavonoid from polygonum aviculare was gotten by extracted with 65% alcohol, gathered with polyamide, and eluted with 75% alcohol. The content of flavone was determined with rutoside as standard preparation. Normal rats thoracic aorta in vitro used as sample, BL-420E biological functional experiment system was utilized to record dilatation effect of total flavonoid on PE affecting pre-contracting blood vessel and the relation between dilatation effect of total flavonoid on blood vessel and calcium influx. Results Total flavonoids from polygonum aviculare can diastole contractions of thoracic aorta caused by PE. In calcium-free perfusion, gradually adding CaCl2 induced calcium influx. Clinical data showed dose-effect relation between drug and blood vessel contraction decreased in the total flavonoids from polygonum aviculare incubation rats than normal rats. Besides, total flavonoids from polygonum aviculare can obviously inhibit contraction of blood vascular circle induced by calcium releasing. Conclusion FP exerted a dose-dependent vasodilatation effect on rat isolated aorta rings by inhibiting Ca2＋ influx via L-type voltage-gated Ca2＋ channels and Ca2＋ release from sarcoplasmic reticulum, consequently decrease Ca2＋ in vascular smooth muscle cells. Key words: Polygonum aviculare; Total flavone; Hypertension; Vasodilation

Calcium signaling of thyrocytes is modulated by TSH through calcium binding protein expression

TSH is an important stimulus to maintain thyroid epithelial differentiation. Impairment of TSH signal transduction can cause thyroid pathologies such as hot nodules, goiter and hyperthyroidism. In a gene expression study in Fischer rat thyroid cells (FRTL-5) using cDNA microarrays we found a TSH-dependent regulation of several calcium binding proteins, S100A4, S100A6 and annexin A6. Expression of these genes in FRTL-5 and regulation by TSH was confirmed with LightCycler qPCR and Western blotting. The differential expression of S100A4 was confirmed for cultured primary human thyrocytes. Calcium-imaging experiments showed that prestimulation with TSH attenuates ATP-elicited P2Y-mediated calcium signaling. Experiments with thapsigargin, TSH and calcium-free perfusion excluded an involvement of other purinergic receptors or an involvement of SERCA regulation. Instead, we find a correlation between S100A4 expression and the effects of TSH on calcium signaling. Overexpression of S100A4 in FRTL-5 and shRNA-mediated knockdown of S100A4 in follicular thyroid cancer cells (FTC133) confirm the ability of S100A4 to attenuate calcium signals. Under repeated stimulations with ATP the calcium retention of these cells is also modulated by S100A4, suggesting a role of S100A4 as calcium buffering protein. As a biological consequence of S100A4 overexpression we detected reduced ATP-stimulated cFos induction. Taken together, the results suggest that S100A4 and other calcium binding proteins are part of a signaling network connecting TSH signaling to calcium-mediated events which play a role in thyroid physiology like H2O2 production or even thyroid cancer.

Endothelium protectant and contractile effects of the antivaricose principle escin in rat aorta

The triterpene saponin escin is the active component of the extract of seeds of Aesculus hippocastanum used in the treatment of chronic venous insufficiency. Escin is also used experimentally to increase membrane permeability in isolated cells. Since endothelial dysfunction is postulated to be involved in venous insufficiency, the possible endothelium-protectant effect of escin was explored in rat aortic rings, a model widely used to study such effects with cardiovascular agents. Escin enhanced endothelium-dependent relaxation induced by acetylcholine when such relaxation had been reduced by exposure to the superoxide ion generator pyrogallol. This effect was attributed to enhanced nitric oxide production by endothelial nitric oxide synthase, a calcium-dependent enzyme, activated by the increased endothelial cell permeability to calcium induced by escin. Another effect of escin thought to contribute to its therapeutic activity is its ability to produce venous contraction. The compound was found to induce concentration-related contraction also in rat aortic rings. This response was partially inhibited by removal of the endothelium or by preincubation with indomethacin, and was completely abolished by incubation in a calcium-free perfusion fluid. Contraction was considered to be due mainly to the aforementioned effect on calcium permeability, with some mediation by release of endothelial vasoconstrictor prostanoids. It was concluded that, in rat aorta, escin possesses an endothelium-protectant action and a direct contractile effect. The former could contribute to its beneficial effect in the treatment of venous insufficiency, while the latter could constitute a limiting side effect.

BDNF Acutely Modulates Synaptic Transmission and Calcium Signalling in Developing Cortical Neurons

Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation(LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca²⁺ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as was the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca²⁺]_i). This effect was partially dependent on extracellular Ca²⁺. In calcium-free perfusion medium a substantial calcium signal remained which disappeared after loading of cortical neurons with 5 µM U-73122. BDNF-induce Ca²⁺ transients were completely blocked by K252a and partially blocked by Cd²⁺. The results demonstrate that BDNF can enhance synaptic transmission and induce directly a rise in [Ca²⁺]_i that require two routes: the release of Ca²⁺ from intracellular calcium stores and influx of extracellular Ca²⁺ mainly through voltage-dependent Ca²⁺ channels in cultured cortical neurons.

Adult ventricular cardiomyocytes: isolation and culture.

Isolated cardiomyocytes are a prerequisite to study the biology of cardiomyocytes. Efficient isolation is difficult, as these cells adhere firmly together in the heart and do not divide. Therefore, any experiment is restricted to the amount of calcium-tolerant, rod-shaped cardiomyocytes that can be initially isolated from the heart. This chapter gives detailed instructions on how ventricular cardiomyocytes can be isolated from an intact adult heart. The method is based on the principle of calcium-free perfusion with collagenase supplementation to disrupt cell-cell contacts in the heart, isolation and purification of cardiomyocytes from other cell types, and, finally, re-establishing a physiological cellular calcium concentration. The chapter also summarizes some commonly used adaptations to isolate cardiomyocytes from species different from rat.

How does chronic manganese chloride application affect the rat isolated ileal contractility?

In this study, we investigated the effects of chronic manganese chloride (MnCl 2 ) application at high dosage on the mechanical responses evoked by acetylcholine (2.7 × 10 -7 M) on isolated rat ileum in standard and calcium-free tyrode perfusion solutions with diltiazem (5.5 × 10 -7 M) and non-diltiazem mediums. In the experimental group (n = 10), MnCl 2 (50 mg/kg/day) was injected for 50 days intraperitoneally. Acetylcholine-induced contraction amplitude increased significantly with respect to that of the control in standard tyrode perfusion medium. When ileal segments were pre-incubated with diltiazem, the amplitude of acetylcholine-induced contractions decreased in experimental and control groups with respect to those of the standard mediums. In calcium-free medium, pretreatment with diltiazem did not cause any change in acetylcholine-induced contractions. Our data suggested that manganese (Mn 2+ ) might have penetrated through cell membrane and accumulated in the cell when manganese was applied chronically and overdose. Increase in plasma manganese level might have induced the increase in manganese influx whereas calcium influx might have been induced by manganese itself. The increase in contraction amplitude may be attributed to this phenomenon.

Effect of ischemia, calcium depletion and repletion, acidosis and hypoxia on cellular taurine content.

Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na(+)](i). The data suggest that taurine efflux is regulated by [Na(+)](i) and cellular osmolality, but not by cellular pH.

Calcium/calmodulin-modulated chloride and taurine conductances in cultured rat astrocytes

Osmotically swollen rat cerebral astrocytes develop an increased anion conductance which can mediate chloride and taurine release. We used whole cell patch clamp to study mechanisms that modulate this conductance. Astrocyte chloride conductance increased within 4 min of exposure to 200 mOsm medium and was 670±123% of its initial value after 15 min (mean±S.E.M.). This conductance was substantially reduced in 0.1 mM extracellular calcium with 20 mM BAPTA added to the electrode solution and was completely inhibited with calcium-free perfusion solution containing 1 mM EDTA ( n=4). The conductance increase in 200 mOsm medium also was inhibited in a dose-dependent manner by nimodipine with a calculated K i of 0.31±0.4 μM and mean±S.E.M. inhibition of 84.4±4% at 100 μM nimodipine. In the presence of 100 μM W-7, a calmodulin antagonist, the mean±S.E.M. conductance increase after 15 min was 223±40% of the initial value while 300 μM W-7 or 100 μM trifluoperazine inhibited the conductance increase completely ( n=6). With taurine as the major anion in electrode and perfusion solutions, a significant conductance increase was observed in 200 mOsm medium. This conductance increase was inhibited by 300 μM W-7 or 100 μM nimodipine. We conclude extracellular calcium influx via L-type calcium channels leads to increased astrocyte anion conductance in 200 mOsm conditions via calmodulin-dependent activation of anion channels. Efflux of anionic taurine from swollen astrocytes also may be affected by calcium influx through a similar calcium/calmodulin-dependent process.

Is cytotoxic cellular edema real? The effect of calcium ion on water homeostasis in the rat heart.

The objective of this study was to determine the effect of calcium on water homeostasis in cardiac muscle and whether cytotoxic edema is involved in ischemic-reperfusion injury. Cellular water was quantified by multinuclear nuclear magnetic resonance (NMR) spectroscopy in isolated rat hearts during 30 min of ischemia and reperfusion. An increase in extracellular calcium from 0.75 to 2.25 mM increased ischemic swelling twofold, with a marginal effect on functional recovery at reperfusion. Inhibition of Ca2+ channels with verapamil and bepridil reduced ischemic swelling by 58-66% and improved cardiac viability and functional recovery. Inhibition of the Na/Ca exchange with amiloride resulted in reperfusion swelling resulting from the inhibition of calcium efflux via the Na/Ca exchange. This was confirmed by inhibition with amiloride of 63% of cellular swelling induced by calcium overload via the Na/Ca exchange after calcium-free perfusion (calcium paradox). The volume-related activity of amiloride was attributed to the inhibition of Na/Ca exchange because the inhibition of the Na/H antiport resulted in low cellular volumes. The consistent changes in cellular volumes induced by the various agents used to alter calcium fluxes provide evidence of a pro-edematous effect of intracellular calcium accumulation during myocardial ischemia. The administration of toxic doses of bepridil to the perfused and ischemic heart resulted in the shrinkage of cellular volumes and in functional failure. The toxicity of bepridil was mediated by unrestricted release of calcium from the sarcoplasmic reticulum because magnesium cardioplegia, which limits calcium release, lowered ischemic swelling without cellular shrinkage and improved functional recovery. In all experimental groups, cellular edema subsided during the early stages of reperfusion, indicating efficient dissipation of intracellular hyperosmolarity. There was no evidence that cytotoxic edema develops during ischemia or reperfusion, or both. On the contrary, calcium-mediated cardiac toxicity was associated with cellular volume shrinkage as a result of the efflux of osmolytes through permeabilized sarcolemmal membranes. It is concluded that calcium ion is involved in the regulation of cellular volumes, and its intracellular accumulation promotes swelling in ischemic myocytes with intact membranes.

Effects of insulin-like growth factor I on basal and stimulated glucose fluxes in rat liver.

Effects of insulin-like growth factor I (IGF-I) and insulin on glucose and potassium fluxes were examined by measuring transhepatic glucose and potassium balance in isolated perfused rat livers. At 1 nM, both IGF-I and insulin decreased basal glucose release by approximately 64% (P < 0.05). Adrenaline (epinephrine)-stimulated glucose release (42.6 +/- 4.5 micromol/g of liver within 30 min) was inhibited (P < 0.05) by approximately 32 and approximately 52% during IGF-I and insulin exposure, which was accompanied by reduced cAMP release (-71 and -80%, P < 0.05). IGF-I- and insulin-induced reduction of glucose release only decreased during calcium-free perfusion, but not during inhibition of phosphoinositide 3-kinase by wortmannin. Both IGF-I and insulin induced net potassium uptake, while insulin also attenuated the response to adrenaline. In conclusion, IGF-I causes (i) insulin-like inhibition of hepatic glycogenolysis, even at low, nanomolar concentrations, which is associated with decreased cAMP release, reduced in the absence of Ca(2+), but not mediated by phosphoinositide 3-kinase, (ii) reduction of adrenaline-induced glycogenolysis and (iii) net potassium uptake under basal conditions.

Effects of insulin-like growth factor I on basal and stimulated glucose fluxes in rat liver

Effects of insulin-like growth factor I (IGF-I) and insulin on glucose and potassium fluxes were examined by measuring transhepatic glucose and potassium balance in isolated perfused rat livers. At 1nM, both IGF-I and insulin decreased basal glucose release by ≈ 64% (P &amp;lt; 0.05). Adrenaline (epinephrine)-stimulated glucose release (42.6±4.5µmol/g of liver within 30 min) was inhibited (P &amp;lt; 0.05) by ≈ 32 and ≈ 52% during IGF-I and insulin exposure, which was accompanied by reduced cAMP release (-71 and -80%, P &amp;lt; 0.05). IGF-I- and insulin-induced reduction of glucose release only decreased during calcium-free perfusion, but not during inhibition of phosphoinositide 3-kinase by wortmannin. Both IGF-I and insulin induced net potassium uptake, while insulin also attenuated the response to adrenaline. In conclusion, IGF-I causes (i) insulin-like inhibition of hepatic glycogenolysis, even at low, nanomolar concentrations, which is associated with decreased cAMP release, reduced in the absence of Ca2+, but not mediated by phosphoinositide 3-kinase, (ii) reduction of adrenaline-induced glycogenolysis and (iii) net potassium uptake under basal conditions.

Oxidative status and anti-oxidant enzyme activity during calcium paradox in the rat isolated heart.

1. The effect of calcium paradox on oxidative status and the activity of anti-oxidant enzymes were studied in the rat isolated heart. Glutathione status, sulphydryl group contents and lipid peroxidation in the myocardium, as well as the release of oxidized and reduced glutathione from the heart, were taken as indices of oxidative events. 2. Reperfusion with calcium after calcium-free perfusion induced a significant decrease in the myocardial content of reduced and oxidized glutathione and non-protein sulphydryl groups. At the same time, a significant release of both forms of glutathione from the heart was observed. However, the ratio of oxidized to reduced glutathione remained unchanged and was not different from control. Increased lipid peroxidation was observed only after 30 min of reperfusion with calcium. 3. Increased anti-oxidant activity during the reperfusion period was observed. Mitochondrial Mn-superoxide dismutase (SOD) activity was increased throughout the reperfusion period, while cytoplasmic Cu,Zn-SOD and glutathione peroxidase activity showed a transient increase at 5 min reperfusion. 4. The results do not support an important role of oxygen free radicals in cell damage observed during calcium paradox in the rat isolated heart. Production of oxygen free radicals may occur during the reperfusion period, but the quantity produced is insufficient to exceed the anti-oxidant capacity of the heart.

Damaging effects of the calcium paradox are reduced in isolated hearts from ethanol-dependent rats: paradoxic effects of dihydropyridine drugs.

Previous experiments showed that isolated hearts from ethanol-exposed rats show a marked increase in sensitivity to anoxic myocardial damage, and we suggested that this may be due to excess calcium entry through L-type voltage-operated calcium channels (L-VOCCs). To challenge this hypothesis, we investigated the effect of ethanol treatment ex vivo on a damaging stimulus, the "calcium paradox," which is associated with removal of calcium from the perfusate. Adult male Sprague-Dawley rats were exposed to intoxicating concentrations of ethanol for 6-10 days by inhalation. Isolated hearts from these animals were perfused with Krebs-Henseleit buffer by using a modified Langendorff technique, and the calcium paradox induced by a 10-min period of perfusion with calcium-free buffer, followed by reperfusion with calcium-containing buffer. Compared with controls, hearts from ethanol-exposed rats were significantly protected against myocardial damage, as shown by a marked reduction in release of intracellular proteins (lactate dehydrogenase, creatine phosphokinase, and myoglobin) during the reperfusion phase. These indices of myocardial damage were modified by the presence of the dihydropyridine (DHP) calcium channel antagonist nitrendipine (10(-6) M) and the DHP L-VOCC activator Bay K 8644 (10(-7) M) in the perfusate during the calcium paradox. Paradoxically, both drugs appeared to increase the damaging effects of calcium-free perfusion, with this effect being generally greater in the preparations from ethanol-exposed rats. As a result, the difference between these hearts and those from control rats was reduced, although a significant degree of protection against the calcium paradox remained. The results support the hypothesis that long-term exposure to ethanol in vivo produces marked alterations in the toxic effects of changes in myocardial calcium concentration. The increased sensitivity to DHP drugs of isolated hearts from ethanol-treated rats is consistent with previous experiments showing increased DHP radioligand-binding sites in these tissues.

Effects of local administration of L-, N-, and P/Q-type calcium channel blockers on spontaneous dopamine release in the striatum and the substantia nigra: a microdialysis study in rat.

The pivotal role for voltage-sensitive calcium channels in initiating synaptic transmitter release is undisputed, but it is only partly known to what extent the different subtypes contribute in vivo. Their importance for the dendritic release of dopamine has not been investigated in vivo previously. To evaluate comprehensively the relative importance of different voltage-sensitive calcium channel subtypes for striatal dopamine release, and to further investigate the mechanism of dendritic dopamine release in the reticulate part of substantia nigra, dopamine was measured by in vivo microdialysis in the striatum or substantia nigra of awake rats. The calcium channel blockers nimodipine, omega-conotoxin-GVIA, omega-agatoxin-IVA, and neomycin were administered locally through the dialysis probes and compared with calcium-free perfusion. Results indicate that dopamine release in the striatum is mainly dependent on N- and P/Q-type channels, but the dendritic dopamine release in the substantia nigra is mediated mainly by some other calcium-dependent mechanism, for example, calcium mobilization through T-, O-, or R-type calcium channels. A portion of the dendritic release is calcium independent but can be inhibited partially by neomycin, which might suggest a role for inositol 4,5-bisphosphate breakdown products.

Release of cerebral 5-hydroxytryptamine evoked by electrical stimulation of the dorsal and median raphe nuclei: effect of a neurotoxic amphetamine.

Recent neuroanatomical data suggest that the axons and terminals of serotonergic neurons of the dorsal and median raphe nuclei are morphologically and pharmacologically distinct. Here we attempted to establish a functional in vivo model of serotonergic terminals derived from these nuclei, and then carry out a preliminary comparison of their physiological and pharmacological properties. Brain microdialysis was used to monitor extracellular 5-hydroxytryptamine in the hippocampus (dorsal and median raphe innervation) and frontal cortex (preferential dorsal raphe innervation) of the anaesthetized rat. To distinguish 5-hydroxytryptamine released by terminals of dorsal raphe neurons from that released by median raphe neurons, one or other of these nuclei was stimulated electrically. Electrical stimulation of either the dorsal or median raphe nucleus evoked a release of 5-hydroxytryptamine in the hippocampus. Whereas stimulation of the dorsal raphe nucleus also released 5-hydroxytryptamine in the frontal cortex, stimulation of the median raphe nucleus did not. No release of 5-hydroxytryptamine was evoked when electrodes were located in regions bordering the dorsal raphe nucleus and the median raphe nucleus. The amounts of hippocampal 5-HT released by stimulation of the dorsal or median raphe nucleus were found to be similarly altered by a 5-hydroxytryptamine uptake inhibitor (citalopram) and calcium-free perfusion medium, and also by increasing stimulation frequency (2-10 Hz). Furthermore, the amount of 5-hydroxytryptamine released by electrical stimulation of either the dorsal raphe nucleus or median raphe nucleus was markedly reduced in rats pretreated with p-chloroamphetamine. In summary, our data show that electrical stimulation of the dorsal or median raphe nucleus releases 5-hydroxytryptamine in a regionally specific manner (hippocampus versus frontal cortex), suggesting that serotonergic nerve terminals of the dorsal and median raphe pathways were being activated selectively. Using this model, we found no differences in the responsiveness of dorsal and median raphe pathways to a specific set of physiological and pharmacological manipulations. In particular, our data suggest that the neurotoxic action of p-chloroamphetamine may not be targeted solely on serotonergic axons and terminals of the dorsal raphe nucleus but includes those of the median raphe nucleus.

Circulatory models in assessment of cardiac enzyme release in dogs.

Cardiac ischemia causes interstitial leakage of cellular enzymes followed by release of these enzymes into plasma. Quantitative interpretation of these data requires a specific circulatory model, and the performance of such models was investigated. Plasma activities of cardiac enzymes were measured for increasingly abrupt forms of ischemic heart injury in the dog: 1) permanent ligation of the left anterior descending coronary artery (LAD); 2) reperfusion after 2 h of ligation of the LAD; and 3) calcium-free perfusion of the LAD during 10 min (calcium-paradox injury). Release into plasma of a rapidly (41%/h) and a slowly (2.2%/h) catabolized enzyme was calculated from the plasma activities, using a detailed circulatory model with compartments for heart, plasma, muscle, skin, and viscera. The time course of cellular enzyme leakage into interstitial space in the heart was calculated from release into plasma and a range of reported values for transendothelial permeability. Simplification to one- and two-compartment models introduced, respectively, 10 and 2% error in calculated cumulative release. Considering the other sources of error, this implies adequate performance of the two-compartment model. Protein washout from the heart is strongly influenced by expansion of interstitial protein space with dead myocyte volume and depends on the microheterogeneity of necrotic tissue areas. Accelerated release of enzymes into plasma after reperfusion reflects accelerated cellular leakage rather than enhanced washout.

Effect of taurine on intracellular calcium dynamics of cultured myocardial cells during the calcium paradox.

The “calcium paradox” phenomenon, first described by Zimmerman and Hulsman1, occurs when hearts are reperfused with calcium after a short period of calcium-free perfusion. The Ca2+ repletion phase causes irreversible myocardial damage, characterized by reduced electrical activity, extensive ultrastructural damage, depletion of tissue high-energy phosphate content, massive release of intracellular constituents and an increase in cytosolic Na+ and Ca2+2.

Nicotine-induced release of noradrenaline and neuropeptide Y in guinea-pig heart: role of calcium channels and protein kinase C

The role of calcium, calcium influx through calcium channels, and activation of protein kinase C for the nicotine-induced release of noradrenaline and of the sympathetic co-transmitter neuropeptide Y (NPY) was investigated in the guinea-pig isolated perfused heart. In the coronary venous overflow noradrenaline and NPY were determined by high-pressure liquid chromatography and radioimmunoassay, respectively. In the presence of extracellular calcium (1.85 mmol/l) nicotine (1-100 mumol/l) evoked a concentration-dependent overflow of both transmitters with a molar ratio of approximately 1500 (noradrenaline):1 (NPY). The nicotine-induced (100 mumol/l) overflow of noradrenaline and NPY was in a linear manner related (r = 0.79 and 0.90, respectively; p less than 0.05) to the extracellular calcium concentration (0-1.85 mmol/l), and it was prevented by calcium-free perfusion. The L-type calcium channel blocker felodipine (100 nmol/l) did not affect the nicotine-induced (100 mumol/l) transmitter overflow. On the other hand, the neuronal (N-type) calcium channel blockers omega-conotoxin (100 nmol/l) and cadmium chloride (50 mumol/l) reduced the nicotine-induced (100 mumol/l) transmitter overflow to 20% of the control value, suggesting a role of N-type calcium channels in mediating the calcium influx for the nicotine-induced transmitter release. The nicotine-induced (30 mumol/l) overflow of both transmitters was two- to three-fold increased by activation of protein kinase C (phorbol 12-myristate 13-acetate; 100 nmol/l). The transmitter overflow was unaffected by 4 alpha-phorbol 12,13-didecanoate (100 nmol/l), a phorbol ester which does not stimulate protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)