Melatonin restores impaired contractility in aged guinea pig urinary bladder

Abstract:  Urinary bladder disturbances are frequent in the elderly population but the responsible mechanisms are poorly understood. This study evaluates the effects of aging on detrusor myogenic contractile responses and the impact of melatonin treatment. The contractility of bladder strips from adult, aged and melatonin-treated guinea pigs was evaluated by isometric tension recordings. Cytoplasmatic calcium concentration ([Ca²⁺]_i) was estimated by epifluorescence microscopy of fura-2-loaded isolated detrusor smooth muscle cells, and the levels of protein expression and phosphorylation were quantitated by Western blotting. Aging impairs the contractile response of detrusor strips to cholinergic and purinergic agonists and to membrane depolarization. The impaired contractility correlates with increased [Ca²⁺]_i in response to the stimuli, suggesting a reduced Ca²⁺sensitivity. Indeed, the agonist-induced contractions in adult strips were sensitive to blockade with Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), but these inhibitors had negligible effects in aged strips. The reduced Ca²⁺ sensitivity in aged tissues correlated with lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and with the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. Interestingly, melatonin treatment restored impaired contractility via normalization of Ca²⁺ handling and Ca²⁺ sensitizations pathways. Moreover, the indoleamine restored age-induced changes in oxidative stress and mitochondrial polarity. These results suggest that melatonin might be a novel therapeutic tool to palliate aging-related urinary bladder contractile impairment.     

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.     

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 .     

Melatonin, as an adjuvant-like agent, enhances platelet responsiveness

Abstract:  Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca²⁺ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca²⁺ release and entry and further increased Ca²⁺ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Melatonin prevents oxidative stress-mediated mitochondrial permeability transition and death in skeletal muscle cells

Abstract:  Oxidative stress-induced mitochondrial dysfunction plays a crucial role in the pathogenesis of a wide range of diseases including muscle disorders . In this study, we demonstrate that melatonin readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptosis of primary muscle cultures prepared from C57BL/6J mice. In particular, melatonin (10⁻⁴–10⁻⁶  m ) fully prevented myotube death induced by tert -butylhydroperoxide ( t -BHP; 10 μ m –24 hr) as assessed by acid phosphatase, caspase-3 activities and cellular morphological changes. Using fluorescence imaging, we showed that the mitochondrial protection provided by melatonin was associated with an inhibition of t -BHP-induced reactive oxygen species generation. In line with this observation, melatonin prevented t -BHP-induced mitochondrial depolarization and mitochondrial permeability transition pore (PTP) opening. This was associated with a highly reduced environment as reflected by an increased glutathione content and an increased ability to maintain mitochondrial pyridine nucleotides and glutathione in a reduced state. Using isolated mitochondria, in a similar manner as cyclosporin A, melatonin (10⁻⁸–10⁻⁶  m ) desensitized the PTP to Ca²⁺ and prevented t -BHP-induced mitochondrial swelling, pyridine nucleotide and glutathione oxidation. In conclusion, our findings suggest that inhibition of the PTP essentially contributes to the protective effect of melatonin against oxidative stress in myotubes.     

Protection against cell death and sustained tyrosine hydroxylase phosphorylation in hydrogen peroxide- and MPP+-treated human neuroblastoma cells with melatonin

Abstract:  Neuroprotective effects of melatonin against oxidative stress-induced neuronal cell degeneration in human SH-SY5Y neuroblastoma cells were investigated in this report. The results demonstrate that exogenous administration of H₂O₂ and 1-methyl, 4-phenyl, pyridinium ion (MPP⁺) significantly decreased cell viability in SH-SY5Y cultured cells. Desipramine, a monoamine uptake blocker was able to abolish the toxic effects of MPP⁺ but not H₂O₂ in reduction of cell viability. Conversely, melatonin reversed the toxic effects of H₂O₂ and MPP⁺ on cell viability. In addition, the reduction of phosphorylation of tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis, and phosphorylation of cyclic AMP responsive element-binding protein by H₂O₂ and MPP⁺ was also diminished by melatonin. These results demonstrate some effective roles of melatonin on neuroprotection and its action on the modulation of tyrosine hydroxylase phosphorylation.     

Bencyclane as an anti-sickling agent

A vasodilating Ca²⁺ channel blocker, bencyclane, was used in 18 patients with homozygous sickle cell anaemia (SCD) to test the possible anti-sickling effect. With bencylane intervention the Na⁺-K⁺ ATPase activity increased from 256±29 to 331±37 nmol Pi/mg protein/h ( P <0.0001) and the Ca²⁺-Mg²⁺ ATPase level increased from 172±12 to 222±44 nmol Pi/mg protein/h ( P <0.0001). The intracytoplasmic Ca²⁺ concentration reduced from 3.5±0.6 to 2.7±0.25 μmol/l ( P <0.0001). The patient's blood contained fewer irreversibly sickled cells (ISCs) (a reduction from 21.4% to 14.4%) ( P <0.05). At the same time MCHC of the erythrocytes decreased from 34.5 to 33.0 g/dl ( P <0.05). Bencyclane appears to be a promising anti-sickling agent that can be used orally in SCD.     

Role of L-Type Calcium Channel Window Current in Generating Current-Induced Early Afterdepolarizations

Ionic Mechanism of EADs. Introduction: Early afterdepolarizations (EADs) can give rise to triggered activity and thereby produce cardiac arrhythmias. We used the whole-cell patch clamp technique to examine the relationship between L-type Ca²⁺ channel window current and the generation of EADs in single ventricular myocytes isolated from guinea pig hearts.
Methods and Results: With a high concentration of EGTA in the internal solution and Na⁺-containing physiologic external solution, EADs were induced in unclamped cells by injecting intracellular depolarizing current pulses. During voltage clamp protocols designed to simulate action potentials interrupted by EADs, we recorded an inward shift in total current up to 0.7 pA/pF over 400 msec at test steps in the range of the take-off potential for EADs. Cd^2t (0.2 mM) blocked most of the inward shift of current during the test steps and abolished EADs. When the same voltage clamp protocol was used following perfusion with an Na⁺-free, K⁺-free external solution, the Cd²⁺-sensitive inward currents recorded during the test steps were similar to those obtained in physiologic external solution. The overlapping range of potentials for partial activation of the d and f variables of L-type Ca²⁺ current ("window" region) measured in Na⁺-free, K⁺-free external solution was virtually the same as the voltage range of the Cd²⁺–sensitive inward currents.
Conclusion: Our experiments suggest that: (1) EADs can arise under conditions of high EGTA buffering of intraccllular [Ca²⁺]; and (2) under these conditions, L-type Ca²⁺ channel window current plays a major role in the initiation of EADs.     

Human Neutrophil Functions Are Inhibited In Vitro by Clinically Relevant Ethanol Concentrations

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs—chemotaxis and production of reactive oxygen species—and on changes in cytosolic-free calcium ([Ca²⁺]_i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b), fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca²⁺]_i, as assessed by videomicroscopy using the Ca²⁺-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca²⁺]_i, levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca²⁺ influx or in [Ca²⁺]_i.     

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.     

Do HbSS erythrocytes lose KCl in physiological conditions?

KCl cotransporter activity in sickle (HbSS) red blood cells (RBCs) was measured in cells suspended in 'simple' physiological saline, saline augmented with inorganic salts, and autologous plasma. Our results showed that the transporter was only functioning at 20% of the level of cells in saline when cells were resuspended in autologous plasma. Kinetic analysis of the data showed that plasma decreased both V _max and K _m for K⁺ of the transporter. The plasma factor(s) responsible was heat-stable and dialysable (i.e. size &60; 10 kD).
Adding magnesium, calcium, inorganic phosphate or bicarbonate to 'simple' saline to mimic the effect of plasma revealed that Mg²⁺ and Ca²⁺ had no significant effect at physiological concentrations. P_i was not effective at 1.1 m M  , but did inhibit significantly (42±2%) at 5.6 m M  . HCO⁻₃   had a major inhibitory effect on K⁺ influx when added to saline, and was identified as the principal candidate for the plasma effect.
We suggest bicarbonate may play a significant role in modifying KCl cotransport, and hence HbSS cell volume in vivo . It acts by altering the set point of the transporter via the signalling systems involved in its regulation.