In vivo and in vitro effects of the pineal gland and melatonin on [Ca2++ Mg2+]-dependent ATPase in cardiac sarcolemma

Abstract: The possible diurnal variation in cardiac [Ca²⁺+ Mg²⁺]-dependent ATPase (Ca²⁺ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemma] membrane [Ca²⁺+ Mg²⁺]-dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time-dependent changes in [Ca²⁺+ Mg²⁺]-dependent ATPase activity did not occur in either pinealectomized or light-exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity. In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca²⁺ pump. The half-maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca²⁺+ Mg²⁺]-dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.     

Restitution of Ca2+ Release and Vulnerability to Arrhythmias

New information has recently been obtained along two essentially parallel lines of research: investigations into the fundamental mechanisms of Ca²⁺-induced Ca²⁺ release (CICR) in heart cells, and analyses of the factors that control the development of unstable rhythms such as repolarization alternans. These lines of research are starting to converge such that we can begin to understand unstable and potentially arrhythmogenic cardiac dynamics in terms of the underlying mechanisms governing not only membrane depolarization and repolarization but also the complex bidirectional interactions between electrical and Ca²⁺ signaling in heart cells. In this brief review, we discuss the progress that has recently been made in understanding the factors that control the beat-to-beat regulation of cardiac Ca²⁺ release and attempt to place these results within a larger context. In particular, we discuss factors that may contribute to unstable Ca²⁺ release and speculate about how instability in CICR may contribute to the development of arrhythmias under pathological conditions.     

The L-type Ca2+ channel is involved in microvesicle-mediated glutamate exocytosis from rat pinealocytes

Abstract: Pinealocytes, parenchymal cells of the pineal gland, secrete glutamate through microvesicle-mediated exocytosis upon depolarization by KC1 in the presence of Ca²⁺, which is involved in a novel paracrine-like intercellular signal transduction mechanism in neuroendocrine organs. In the present study, we investigated whether or not the L-type Ca²⁺ channel is involved in the microvesicle-mediated glutamate secretion from cultured rat pinealocytes. Nifedipine, a specific antagonist of the L-type Ca²⁺ channel, inhibited the Ca²⁺-dependent glutamate exocytosis by 48% at 20 uM. Other L-type Ca²⁺ channel antagonists, such as nitrendipine, showed similar effects. 1,4-Dihydro-2,6-dimethyl-5-nitro-4[2-(trifluoromethyl)-phenyl]-3-pyridinecarboxylic acid methyl ester (BAY K8644), an agonist of the L-type Ca²⁺ channel, at 1 uM, on the other hand, stimulated the glutamate exocytosis about 1.6-fold. Consistently, these Ca²⁺ channel antagonists inhibited about 50% of the Ca²⁺ uptake, whereas BAY K8644 increased the uptake 5.3-fold. An antibody against the carboxyl-terminal region of the rabbit L-type Ca²⁺ channel recognized polypeptides of pinealocytes with apparent molecular masses of 250 and 270 kDa, respectively, and immunostained the plasma membrane region of the pinealocytes. These results strongly suggested that the entry of Ca²⁺ through L-type Ca²⁺ channel(s), at least in part, triggers microvesicle-mediated glutamate exocytosis in pinealocytes.     

Effect of Chronic Ethanol Feeding on Kupffer Cell-Mediated Antitumor Cell Activity

We have previously reported that the Kupffer cell has antitumor activity through mitochondrial damage to tumor cells by nitric oxide production. In this study, the effect of chronic ethanol feeding on antihepatoma cell activity of the Kupffer cell was examined in rats. Male rats of the Wistar strain were fed ethanol chronically for 8 weeks by liquid diets. Kupffer cells were isolated from the control rat or the ethanol-fed rat, and cocultured with AH 70 cells, a rat hepatoma cell line. Fluorescence of rhodamine 123 or propidium iodide was observed as indicators of the mitochondrial damage or cell membrane injury, respectively, by a laser scanning confocal microscopy. Mitochondrial damage of AH 70 cells as indicated by reduction of rhodamine 123 fluorescence was smaller by the coculture with Kupffer cell from the ethanol rat than that from the control. Cell membrane barrier dysfunction of AH 70 cell was less frequently observed with the Kupffer cell from ethanol-fed rats. A metabolite of nitric oxide (nitrite and nitrate) was less in the cultured medium with the ethanol Kupffer cell than with the control Kupffer cell. Ca²⁺ mobilization, which induces inducible nitric oxide synthase and observed by the fluorescence of fluo-3, in Kupffer cells cocultured with AH 70 cells was suppressed in ethanol-fed rats. These result suggests that chronic ethanol feeding suppresses antitumor cell activity of Kupffer cell through the impairment of Ca²⁺ mobilization and nitric oxide production.     

Protective effect of melatonin on Ca2+ homeostasis and contractility in acute cholecystitis

Abstract:  Impaired Ca²⁺ homeostasis and smooth muscle contractility co-exist in acute cholecystitis (AC) leading to gallbladder dysfunction. There is no pharmacological treatment for this pathological condition. Our aim was to evaluate the effects of melatonin treatment on Ca²⁺ signaling pathways and contractility altered by cholecystitis. [Ca²⁺]_i was determined by epifluorescence microscopy in fura-2 loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were determined by spectrophotometry and cycloxygenase-2 (COX-2) expression was quantified by western blot. Melatonin was tested in two experimental groups, one of which underwent common bile duct ligation for 2 days and another that was later de-ligated for 2 days. Inflammation-induced impairment of Ca²⁺ responses to cholecystokinin and caffeine were recovered by melatonin treatment (30 mg/kg). This treatment also ameliorated the detrimental effects of AC on Ca²⁺ influx through both L-type and capacitative Ca²⁺ channels, and it was effective in preserving the pharmacological phenotype of these channels. Despite its effects on Ca²⁺ homeostasis, melatonin did not improve contractility. After de-ligation, Ca²⁺ influx and contractility were still impaired, but both were recovered by melatonin. These effects of melatonin were associated to a reduction of MDA levels, an increase in GSH content and a decrease in COX-2 expression. These findings indicate that melatonin restores Ca²⁺ homeostasis during AC and resolves inflammation. In addition, this indoleamine helps in the subsequent recovery of functionality.     

Na+/Ca2+ Exchanger Expression and Function in a Rabbit Model of Myocardial Infarction

Introduction: In general, sarcolemmal Na⁺/Ca²⁺ exchanger (NCX) protein and activity is increased in hearts with ventricular dysfunction. However, in a subset of studies, reduced activity of NCX has been reported. Left ventricular dysfunction (LVD) was induced in the rabbit eight weeks after an apical myocardial infarction.
Methods: Using single microelectrode voltage clamp to assess the NCX activity in isolated ventricular cells, a decrease in NCX activity by ∼30% was observed. Immunoblot analysis indicated increased NCX protein levels by ∼20% in the LVD group. The cause of this paradox is unknown. Overexpression of the protein sorcin increased the activity of NCX without affecting NCX protein levels.
Results: Sorcin protein (dimer) levels were significantly lower in the LVD group (0.67 ± 0.05 n = 15, P < 0.05) compared to sham (1.0 ± 0.16, n = 15). Sorcin monomer levels were not significantly different (sham: 1.0 ± 0.26, LVD: 0.83 ± 0.13). Mathematical modeling of NCX suggests that a reduction of NCX activity during diastole to that in LVD could be achieved by holding the diastolic membrane potential at −60 mV instead of −80 mV. Holding E_m at −60 mV decreased NCX-mediated Ca²⁺ efflux rates to values comparable to those seen in LVD and increased SR Ca²⁺ content and peak systolic [Ca²⁺] in sham and LVD cardiomyocytes.
Conclusions: In conclusion, reduced sorcin expression may be linked to the lower NCX activity in the rabbit model of LVD. Reduced NCX activity during diastole increases SR Ca²⁺ content and Ca²⁺ transient amplitude.     

A requirement for calcium in the caspase-independent killing of Burkitt lymphoma cell lines by Rituximab

The therapeutic monoclonal antibody rituximab has previously been shown to kill B cells in a caspase-independent manner. The signalling pathways underpinning this novel death pathway are unknown. The present study showed that rituximab treatment of Burkitt lymphoma cell lines induced a slow rise in intracellular calcium ([Ca²⁺]_i). This rise was only witnessed in cell lines that were killed by antibody, suggesting a critical role for Ca²⁺ in mediating rituximab-driven caspase-independent cell death. Inhibition of the two main intracellular store-located Ca²⁺ channels, i.e. the ryanodine and inositol-1,4,5-triphosphate receptor channels by dantrolene and xestospongen-c respectively did not prevent the rise in Ca²⁺ seen with rituximab or protect cells from subsequent death. In sharp contrast, inhibition of Ca²⁺ entry via plasma membrane channels with (2-aminoethoxy) diphenylborate or SKF-96365 or complete chelation of extracellular Ca²⁺ with ethyleneglycol bis (aminoethylether) tetra-acetate inhibited the rise in [Ca²⁺]_i and increased cell viability. Together, these data suggest that ligation of the CD20 receptor with rituximab allows a slow sustained influx of Ca²⁺ from the external environment that under certain conditions can lead to cell death.     

Abnormal cAMP-induced phosphorylation of rap 1, protein in grey platelet syndrome platelets

Summary We previously demonstrated abnormal Ca²⁺ transport by microsomes in platelets from a grey platelet syndrome patient. Here, we investigated the platelet Ca²⁺ ATPases that mediate this transport, as well as its possible regulation by rap 1 protein. We showed that grey platelet syndrome platelets expressed the same two distinct Ca²⁺ ATPases as those recently described in normal platelets; the 100 kD SERCA_2-b isoform (Sarco/Endoplasmic Reticulum Ca²⁺ATPase) and a new 97 kD SERCA isoform. The two Ca²⁺ ATPases formed similar amounts of transient phosphorylated intermediates. The expression of these two Ca²⁺ ATPases was compared by Western blotting using specific antibodies, which again emerged in similar amounts in normal and grey platelet syndrome platelets. As regards the protein phosphorylated by cAMP, it was found to be identical to rap 1 protein when it was immunoprecipitated with an antibody raised against a synthetic peptide specific for rap 1 protein. Although the expression of rap 1 protein was similar in membranes isolated from grey platelet syndrome and normal platelets, its exogenous phosphorylation by cAMP was abnormal, with a concentration (10 μg/ml) of the catalytic subunits of the cAMP-dependent protein kinase (C.Sub.), as it decreased to half the control level.
It is concluded that the abnormal Ca²⁺ transport found in grey platelet syndrome platelets is not due to the abnormal expression of the Ca²⁺ ATPases, but is associated with an abnormality of rap 1 protein phosphorylation by cAMP.     

Age-related alterations in Ca2+ signals and mitochondrial membrane potential in exocrine cells are prevented by melatonin

Abstract:  Information regarding age-induced Ca²⁺ signal alterations in nonexcitable cells is limited. In addition, little evidence exists on the ability of melatonin to palliate the effects of aging on Ca²⁺ signals and mitochondrial potential, a parameter involved in both Ca²⁺ signaling and aging. We studied the ability of melatonin to prevent the effects of aging on intracellular Ca²⁺ homeostasis and mitochondrial potential in exocrine cells. Pancreatic acinar cells were obtained from adult (3 months old) and aged (22–24 months old) mice by collagenase dispersion. Ca²⁺ signals, in situ mitochondrial potential and in vitro amylase secretion were determined. Secretion in response to increasing levels of the secretagogues, acetylcholine and cholecystokinin (CCK), were impaired in aged pancreatic acini. This decrease was accompanied by an inhibition in the amplitude of the peak response to maximal concentrations of the agonists, and by a decrease in the pattern of Ca²⁺ oscillations induced by postprandial levels of CCK. Both the size of the calcium pools, assessed by low levels of ionomycin, and capacitative calcium entry, induced by depletion of the stores with thapsigargin, were diminished in aged cells. These changes in Ca²⁺ homeostasis were associated with depolarization of intracellular mitochondria. Oral administration of melatonin for 3 months to aged mice restored the secretory response, the amplitude and frequency of Ca²⁺ responses, the size of intracellular calcium pools, the capacitative calcium entry, and the mitochondrial potential. In conclusion, melatonin restores secretory function, Ca²⁺ signals and mitochondrial potential of aged exocrine cells.     

Action Potential Duration, Rate of Stimulation, and Intracellular Sodium

In the first section of this short review the change of the cardiac action potential (APD) with the rate of stimulation under physiological conditions is described and mechanistically analyzed. A fast phase of adaptation is mainly caused by changes in gating characteristics of ionic currents, and rapid modulation of the Na⁺/Ca²⁺ exchanger. The slower phase is largely conditioned by incomplete recovery from inactivation of the late Na⁺ current (late I_Na) and changes in ion concentrations of [K⁺]_e, [Na⁺]_i, and [Ca²⁺]_i, which cause secondary changes in the permeation and the gating of ion channels and flux through transporters. In a second section, an analysis is presented of the rate dependence of APD in pathological conditions and its importance in the genesis of arrhythmias in hypertrophy, heart failure, congenital, and acquired LQT syndromes is summarized. The role of the late I_Na, Na⁺, and Ca²⁺ overload is emphasized. Special attention is given to the paradoxical transient lengthening of APD in LQT3 syndrome for the sudden increase in rate in this setting. The third section consists of a short commentary on Na⁺ and Ca²⁺ overload and drugs which block the late I_Na.     

Intervention of pyrrolidine dithiocarbamate and tetrandrine on cellular calcium overload of pancreatic acinar cells induced by serum and ascitic fluid from rats with acute pancreatitis

Background and Aim:  To investigate the effects of serum and ascitic fluid from rats with acute pancreatitis (AP) on cellular free calcium concentration ([Ca²⁺]i) of isolated rat pancreatic acinar cells, and the intervention of pyrrolidine dithiocarbamate (PDTC) and tetrandrine (Tet) to cellular calcium overload in AP.
Methods:  AP was induced in Sprague–Dawley rats with a retrograde pancreatic duct injection of 3% sodium deoxycholate, and confirmed by histopathological examination and amylase activity assay. The rat serum and ascitic fluid were collected at 1, 5 and 10 h after AP induction, and used as irritants on isolated rat pancreatic acinar cells. The effects on intracellular [Ca²⁺]i, and cell viability were examined. Then, the antagonistic effects of different concentrations of PDTC and Tet were assessed.
Results:  The irritation with AP serum and ascitic fluid reduced the survival rate of the isolated rat pancreatic acinar cells and increased the cellular [Ca²⁺]i significantly ( P  < 0.05). As AP induction course prolonged, the stimulation effect of the AP serum and ascitic fluid intensified. In the pretreated acinar cells with PDTC or Tet, the decreased cell vitality reverted. The elevation of [Ca²⁺]i in the acinar cells significantly ameliorated (significant, P  < 0.05; very significant, P  < 0.01).
Conclusion:  The serum and ascitic fluid from AP rats drastically elevate the [Ca²⁺]i in isolated pancreatic acinar cells and decrease cell vitality, while the pretreatment of cells with PDTC and Tet offsets the calcium overload irritated by the AP serum and ascitic fluid and protects these isolated acinar cells.     

Von Willebrand factor protects the Ca2+-dependent structure of the factor VIII light chain

We have recently reported that cation-exchange iminodiacetate resin completely inactivated factor VIII (FVIII) by direct deprivation of metal ions, predominantly Ca²⁺, from its molecules, and that von Willebrand factor (VWF) protected FVIII antigen from resin-induced degradation. The present study was further developed to investigate this mechanism. Western blotting analysis and enzyme-linked immunosorbent assay showed that the antigenic structure of the FVIII light chain, especially the C2 domain, was completely impaired by the resin, whilst that of the heavy chain was little affected. However, the complex formation with VWF protected the C2 domain from the resin-induced degradation. The resin-treated C2 domain failed to interact with VWF and phospholipid. Furthermore, the addition of Ca²⁺ competitively blocked the resin-induced impairment of the C2 domain structure. These results demonstrate that VWF protects the Ca²⁺-dependent conformational structure of the FVIII light chain, especially the C2 domain, and may indicate that the C2 domain contains the Ca²⁺-binding site(s).     

Ethanol and Cocaine Cause Additive Inhibitory Effects on the Calcium Transients and Contraction in Single Cardiomyocytes

The heart is a major locus for the toxic actions of cocaine and ethanol, each of which has been shown to interfere with excitation-contraction coupling in cardiac muscle cells. Because these drugs are frequently used in combination, the present study was designed to investigate how they interact to modify the Ca²⁺ transient and associated contraction in fura2-loaded cardiomyocytes. A high-speed imaging technique using a charge-coupled device as detector and short-term image store was used to measure cytosolic Ca²⁺ and contraction simultaneously from fluorescence images obtained during the contractile cycle. Ethanol (100 mM) and cocaine (50 μM) caused reversible reductions in Ca²⁺ transient amplitude of 24.3 ± 3.0% and 25.1 ± 3.6%, respectively. Neither agent modified basal Ca²⁺. Ethanol treatment decreased peak shortening by 44.3 ± 3.5%, whereas the contractile depression by cocaine was 31.4 ± 5.3%. The relatively greater effect of ethanol on contraction resulted from a Ca²⁺-independent component of ethanol action on contractility. When cardiomyocytes were exposed simultaneously to ethanol and cocaine, Ca²⁺ transient amplitude was reduced by 38.7 ± 3.0%, and peak contraction was decreased by 55.1 ± 3.5%. These values represent a significantly greater inhibition than observed with either drug alone (p < 0.02) and are compatible with additive effects of the two drugs acting at distinct loci within the excitation-contraction coupling pathway. Thus, simultaneous use of cocaine and ethanol leads to an enhanced depression of myocardial contractility, which is likely to contribute to the cardiotoxic actions of the combination of these two drugs.     

Altered T-Lymphocyte Responsiveness to Polyclonal Cell Activators Is Responsible for Liver Cell Necrosis in Alcohol-fed Rats

The role of T-cell activation in alcoholic liver disease was investigated in rats fed alcohol and subsequently exposed to concanavalin A (Con A). Following Con A injection (20 mg/kg body weight), greater increases in liver-to-body weight ratio and ALT levels were observed at 12 and 24 hr in rats fed ethanol, compared with control rats fed sucrose. Furthermore, increases in serum interleukin-6 and tumor necrosis factor-α levels were noted in ethanol-fed rats, with maximal levels detected at 4 hr declining thereafter, but remaining above control levels at 24 hr. Analysis of T-cell subpopulations showed an increased percentage of CD4⁺, CD5⁺, and CD8⁺ T cells in blood from all groups, but not in liver perfusate. In contrast, a significant increase in the percentage of activated CD25⁺ T cells was detected in both blood and liver perfusate from rats fed ethanol even 24 hr after Con A injection. When CD4⁺ and CD8⁺ T cells from liver perfusate were cultured in the absence or presence of Con A, an increase in interleukin-6 and tumor necrosis factor-α production in supernatants was observed in ethanol-fed rats. In cultures stimulated with Con A, a 2- to 8-fold increase in cytokine production was detected, with intrahepatic CD4⁺ T cells being the major source. Immunohistological analysis revealed infiltration of CD4⁺ T cells around portal vein and central vein areas associated with fatty liver and severe hepatic necrosis. The results suggest that alcohol consumption induced a dysregulated T-cell population that mediated hepatic necrosis following polyclonal activation with Con A.     

Increased erythropoiesis of β-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation

β - thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of β-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34⁺ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in β-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca²⁺. Modulation of either cAMP or Ca²⁺ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of β-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca²⁺.     

pH-Regulated Na+ Influx into the Mammalian Ventricular Myocyte: The Relative Role of Na+-H+ Exchange and Na+-HCO3− Co-Transport

In the heart, intracellular Na⁺ concentration (Na⁺_i) is a controller of intracellular Ca²⁺ signaling, and hence of key aspects of cell contractility and rhythm. Na⁺_i will be influenced by variation in Na⁺ influx. In the present work, we consider one source of Na⁺ influx, sarcolemmal acid extrusion. Acid extrusion is accomplished by sarcolemmal H⁺ and HCO₃⁻ transporters that import Na⁺ ions while exporting H⁺ or importing HCO₃⁻. The capacity of this system to import Na⁺ is enormous, up to four times the maximum capacity of the Na⁺-K⁺ ATPase to extrude Na⁺ ions from the cell. In this review we consider the role of Na⁺-H⁺ exchange (NHE) and Na⁺-HCO₃⁻co-transport (NBC) in mediating Na⁺ influx into cardiac myocytes. We consider, in particular, the role of NBC, as so little is known about Na⁺ influx through this transporter. We show that both proteins mediate significant Na⁺ influx and that although, in the ventricular myocyte, NBC-mediated Na⁺ influx is less than through NHE, the proportions may be altered under a variety of conditions, including exposure to catecholamines, membrane depolarization, and interference with activity of the enzyme, carbonic anhydrase.     

Shortcomings of current models of glucose-induced insulin secretion

Glucose-induced insulin secretion by pancreatic β-cells is generally schematized by a 'consensus model' that involves the following sequence of events: acceleration of glucose metabolism, closure of ATP-sensitive potassium channels (K_ATP channels) in the plasma membrane, depolarization, influx of Ca²⁺ through voltage-dependent calcium channels and a rise in cytosolic-free Ca²⁺ concentration that induces exocytosis of insulin-containing granules. This model adequately depicts the essential triggering pathway but is incomplete. In this article, we first make a case for a model of dual regulation in which a metabolic amplifying pathway is also activated by glucose and augments the secretory response to the triggering Ca²⁺ signal under physiological conditions. We next discuss experimental evidence, largely but not exclusively obtained from β-cells lacking K_ATP channels, which indicates that these channels are not the only possible transducers of glucose effects on the triggering Ca²⁺signal. We finally address the identity of the widely neglected background inward current (Cl⁻ efflux vs. Na⁺ or Ca²⁺ influx through voltage-independent channels) that is necessary to cause β-cell depolarization when glucose closes K_ATP channels. More attention should be paid to the possibility that some components of this background current are influenced by glucose metabolism and have their place in a model of glucose-induced insulin secretion.     

Microvesicles from Sickle Erythrocytes and their Relation to Irreversible Sickling

S ummary . Incubation of sickle (HbS) erythrocytes for periods up to 96 h leads to the formation of irreversibly sickled cells (ISCs) and to the release of spectrin-free microvesicles similar to those derived from aged or Ca²⁺-ionophore-treated normal erythrocytes. The sickle microvesicles were somewhat larger than those from normal cells and showed minor differences in their membrane polypeptide composition. Sickle microvesicles were no different from their parent cells in their content of fetal haemoglobin. Neither microvesiculation nor formation of irreversibly sickled cells required the presence of Ca²⁺ in the medium but Ca²⁺ did accelerate both processes. Although in these prolonged incubations microvesiculation appeared to occur concomitantly with the formation of ISCs, it is not clear whether or not microvesiculation is a necessary prelude to irreversible sickling.     

Regulation of calcium influx and phospholipase C activity by indoleamines in dinoflagellate Crypthecodiniurn cohnii

Abstract: Exogenous indoleamines such as melatonin and 5-methoxytryptamine have been shown to induce cyst formation (encystment) in many species of dinoflagellate. Induction of inositol phosphates formation by indoleamine has previously been demonstrated in Crypthecodiniurn cohnii . In addition, depletion of extracellular Ca²⁺ blocks the indoleamine-induced encystment. In the present study, 12 indoleamines (including melatonin and related compounds) were examined for their abilities to induce Ca²⁺ influx, inositol phosphates formation, and encystment in C. cohnii . The results showed that melatonin, 5-methoxytryptamine, and the peptide toxin mastoparan stimulated ⁴⁵Ca²⁺ influxes in dose- and time-dependent manners. The EC₅₀ values of 5-methoxytrypramine and mastoparan to stimulate ⁴⁵Ca²⁺ uptake were 2 mM and 35 μM, respectively. The 5-methoxytryptamine- and mastoparan-induced ⁴⁵Ca²⁺ influx were partially attenuated by the calcium channel blockers, verapamil and ruthenium red. A series of indoleamines were examined for their structure-activity relationship on the induction of encystment and formation of inositol phosphates. Melatonin-induced inositol phosphates formation was completely blocked by U73122, indicating the possible involvement of phospholipase C. Taken together, we conclude that indoleamines may induce encystment of the dinoflagellate C. cohnii via parallel activation of phospholipase C and Ca²⁺ influx signaling pathways. However, activation of phospholipase C and Ca²⁺ influx are not always necessary or sufficient for inducing encystment. Also, these data provided the first direct evidence of a Ca²⁺ influx regulating mechanism in dinoflagellate C. cohnii .     

Modulation of Two Cloned Potassium Channels by 1-Alkanols Demonstrates Different Cutoffs

It is not known whether alcohols modulate ion channels by directly binding to the channel protein or by perturbing the surrounding membrane lipid. Cutoff describes the phenomenon where the potency of 1-alkanols monotonically increases with alkyl chain length until a loss of efficacy occurs. Determination of the cutoff for a variety of channels can be important, because similar and/or dissimilar cutoffs might yield information regarding the nature of ethanol's site of action. In this study, the two-electrode voltage clamp technique was used to determine the cutoffs for the 1-alkanol potentiation of cloned Ca²⁺-activated-K⁺ (BK) channels and for the inhibition of cloned Shaw2 K⁺ channels, expressed in Xenopus oocytes. Ethanol, butanol, hexanol, and heptanol reversibly enhanced BK currents, whereas octanol and nonanol had no effect. In contrast, Shaw2 currents were potently inhibited by both octanol and decanol, but not by undecanol. Taken together, data demonstrate that the modulation of K⁺ channels by long chain alcohols is channel-specific. Interestingly, ethanol was a less potent activator of BK currents in the intact oocyte in comparison with its effect on this channel in excised membrane patches. The decrease in potency could not be attributed to an ethanol-dependent change in Ca²⁺ influx through endogenous voltage-gated channels, an effect that would alter the concentration of Ca²⁺ available to activate BK channels.