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.     

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 Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to Endotoxin

Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-α (TNF-α) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-α should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca²⁺]_i) is required for LPS-induced TNF-α production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca²⁺]_i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin.
Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 μmol/liter). After addition of LPS (10 μg/ml) to culture media, [Ca²⁺]_i was measured using a fluorescent indicator, fura-2.
Results: LPS increased [Ca²⁺]_i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 μg/ml)-induced TNF-α production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05).
Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin.     

Platelet activation induced by an antiplatelet autoantibody against CD9 antigen and its inhibition by another autoantibody in immune thrombocytopenic purpura

Summary. In a patient with immune thrombocytopenic purpura (ITP), we found a novel platelet-activating IgG (act-IgG) and an inhibitory IgG (inhi-IgG) that prevented activation induced by both CD9 monoclonal antibody (mAb) and the act-IgG. Purified IgG from the patient plasma caused a rise in [Ca²⁺]_i and the aggregation of normal platelets, and bound to a 24 kD membrane protein. This aggregation was inhibited by aspirin, staurosporine, an inhibitor of protein kinase C, and F(ab')₂ fragments of MALL13, a CD9 mAb. When the platelet count of this patient rose to normal range, the act-IgG disappeared. About 2 weeks later, the relapse of thrombocytopenia was observed. The purified IgG obtained in this period did not activate platelets but inhibited both the rise in [Ca²⁺]_i and platelet aggregation stimulated by NNKY 1–19, a CD9 mAb, as well as the act-IgG, and bound to a 40 kD membrane protein. The inhi-IgG prevented the binding of IV-3, a mAb against Fcγ receptor II (FcγRII), but did not prevent the binding of NNKY 1–19 to its antigen. We suggest that the activating autoantibody recognized CD9 antigen and activated both the thromboxane- and phospholipase C-dependent pathways, while the inhibitory autoantibody recognized the FcγRII and inhibited CD9 antibody-induced platelet activation mediated via this receptor.     

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.     

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.     

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.     

Effects of Chronic Ethanol on the Mobilization of Arachidonate and Docosahexaenoate Stimulated by the Type 2A Serotonin Receptor Agonist (±)-2,5-Dimethoxy-4-iodoamphetamine Hydrochloride in C6 Glioma Cells

We studied the effects of chronic ethanol exposure on the mobilization of polyunsaturated fatty acids stimulated by activation of the type 2A serotonin receptor in C6 glioma cells. In our in vitro model, we prelabeled cells with [³H]arachidonate and [¹⁴C]docosahexaenoate and subsequently stimulated with the type 2A serotonin receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride. In as early as 10 days of exposure to 20 or 50 mM ethanol, the (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride-stimulated mobilization of [³H]arachidonic acid ([³H]AA) and [¹⁴C]docosahexaenoic acid ([¹⁴C]DHA) was significantly inhibited, and this inhibition was accompanied by decreased mobilization of intracellular [Ca²⁺]_i. Exposure to ethanol did not alter significantly the release of [³H]AA and [¹⁴C]DHA stimulated by the calcium ionophore A23187 nor the incorporation of [³H]AA and [¹⁴C]DHA into cellular lipids. Decreased mobilization of polyunsaturated fatty acids and calcium in astroglia may contribute to neurotoxicity caused by chronic ethanol exposure.     

β-cell hyperexcitability: from hyperinsulinism to diabetes

Nutrient oxidation in β cells generates a rise in [ATP]:[ADP] ratio. This reduces K_ATP channel activity, leading to depolarization, activation of voltage-dependent Ca²⁺ channels, Ca²⁺ entry and insulin secretion. Consistent with this paradigm, loss-of-function mutations in the genes ( KCNJ11 and ABCC8 ) that encode the two subunits (Kir6.2 and SUR1, respectively) of the ATP-sensitive K⁺ (K_ATP) channel underlie hyperinsulinism in humans, a genetic disorder characterized by dysregulated insulin secretion. In mice with genetic suppression of K_ATP channel subunit expression, partial loss of K_ATP channel conductance also causes hypersecretion, but unexpectedly, complete loss results in an undersecreting, mildly glucose-intolerant phenotype. When challenged by a high-fat diet, normal mice and mice with reduced K_ATP channel density respond with hypersecretion, but mice with more significant or complete loss of K_ATP channels cross over, or progress further, to an undersecreting, diabetic phenotype. It is our contention that in mice, and perhaps in humans, there is an inverse U-shaped response to hyperexcitabilty, leading first to hypersecretion but with further exacerbation to undersecretion and diabetes. The causes of the overcompensation and diabetic susceptibility are poorly understood but may have broader implications for the progression of hyperinsulinism and type 2 diabetes in humans.     

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.     

Role of the GTP-Binding Protein Go in the Suppressant Effect of Ethanol on Voltage-Activated Calcium Channels of Murine Sensory Neurons

Whole-cell and single-channel recording techniques were used to investigate the acute, in vitro effects of ethanol on the function of voltage-activated Ca²⁺ channels in cultured neurons derived from dorsal root ganglia (DRG) of embryonic mice. Although 5.4 MM ethanol produced a sustained increase of the amplitude of the whole-cell Ca²⁺ current (I_Ca), 43.2 mM ethanol had a time-dependent biphasic effect. That is, within 0.5 min of exposure to 43.2 MM ethanol, the maximal amplitude of I_Ca initially increased before declining to a new steady-state value. As anticipated, the facilitatory and inhibitory effects of ethanol on I_Ca were associated with an increase and decrease, respectively, in the probability of single-channel open events. Pretreatment of DRG with 200 ng/ml of pertussis toxin abolished the inhibitory, but not the facilitatory, effect of 43.2 mM ethanol on I_Ca Pretreatment with pertussis toxin also prevented the reduction of the probability of single-channel opening caused by 43.2 mM ethanol. Similarly, dialysis of neurons with polyclonal antibodies against the a-subunit of Go but not G., abolished the inhibitory effect of 43.2 mM ethanol on I_Ca These data demonstrate concentration- and time-dependent biphasic effects of ethanol on the activity of Ca²⁺ channels. The inhibitory effect of ethanol requires activation of the a-subunit of Go, which then decreases the probability of Ca²⁺ channel opening.     

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.     

Analysis of clinical AIDS-free interval after CD4+ cell counts fall below 200 × 106 L−1 in Japanese Haemophiliacs infected with HIV-1

We analysed the time from the date CD4+ cell counts fell below 200 × 10⁶ L⁻¹, defined as t _i, to the onset of clinical AIDS, according to the 1987 Centers for Disease Control and Prevention case definition, in 129 Japanese Haemophilia patients infected with HIV-1. The cumulative onset of clinical AIDS was analysed by the Kaplan–Meier method and proportional hazard model. Incorporated covariates were age of each patient at time t _i, as well as CD4+ and CD8+ cell counts, serum levels of IgG, IgA, IgM, GOT and GPT at t _i. The time of antiretroviral treatment initiation was also considered. The 50% AIDS-free interval after t _i was 3.00 years (95% confidence interval (CI), range 0.49–5.51) and 1.71 years (95% CI, range 0.66–2.76) for the patients at CDC stage II and stage III, respectively (significantly different, P = 0.0013). Among the patients at CDC stage II at t _i, higher levels of IgA were tightly associated with a shorter period from t _i to onset of clinical AIDS ( P < 0.0001), and relative hazard was 1.35 (95% CI, 1.11–1.64) with increase of IgA level by 1.0 g L⁻¹. Thus there is a broad distribution in the time to onset of clinical AIDS in Japanese Haemophiliacs even after CD4+ cell counts fall below 200 × 10⁶ L⁻¹. This should be taken into consideration in deciding upon the therapy and care of HIV-1 infected people.     

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.     

Function, Regulation, Pharmacology, and Molecular Structure of ATP-Sensitive K+ Channels in the Cardiovascular System

K_ATP Channels in Cardiovascular System. ATP-sensitive K⁺ (K_ATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. K_ATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K_ATP channel appears to be activated during ischemic or hypoxic conditions and may be responsible for the increase of K⁺ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective as well as proarrhythmic effects. In the vascular smooth muscle, the K_ATP channel is believed to mediate the relaxation of vascular tone. Thus, K_ATP channels play important regulatory roles in the cardiovascular system. Furthermore, K_ATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas, which block the channels, and a series of vasorelaxants called "K⁺ channel openers," which tend to maintain the channels in an open conformation. Recently, the molecular structure of K_ATP channels has been clarified. The K_ATP channel in pancreatic β-cells is a complex composed of at least two subunits, a member of inwardly rectifying K⁺ channels and a sulfonylurea receptor. Subsequently, two additional homologs of the sulfonylurea receptor, which form cardiac and smooth muscle type K_ATP channels, respectively, have been reported. Further works are now in progress to understand the molecular mechanisms of K_ATP channel function.     

Quantitative Reconstruction of Cardiac Electromechanics in Human Myocardium:

Introduction: Myocytes from normal and failing myocardium show significant differences in electromechanical behavior. Mathematical modeling of the behavior provides insights into the underlying physiologic and pathophysiologic mechanisms. Electromechanical models of cardiomyocytes exist for various species, but models of human myocytes are lacking.
Methods and Results: A mathematical model of electromechanics in normal and failing cardiac myocytes in humans was created by assembly and adaptation of parameters of an electrophysiologic model at the level of single cells and a force development model at the level of the sarcomere. The adaptation was performed using data from recent studies of ventricular myocytes and myocardium. The model was applied to quantitatively reconstruct measurement data from different experimental studies of normal and failing myocardium. Several simulations were performed to quantify the transmembrane voltage V_m , intracellular concentration of calcium[ Ca²⁺ ] _i , the [ Ca²⁺ ] _i –force relationship, and force transients. Furthermore, frequency dependencies and restitution of action voltage duration to 90% recovery APD₉₀, peak [ Ca²⁺ ] _i , duration to 50% force recovery FD₅₀, and peak force were determined.
Conclusion: The presented mathematical model was capable of quantitatively reconstructing data obtained from different studies of electrophysiology and force development in normal and failing myocardium of humans. In future work, the model can serve as a component for studying macroscopic mechanisms of excitation propagation, metabolism, and electromechanics in human myocardium. (J Cardiovasc Electrophysiol, Vol. 14, pp. S210-S218, October 2003, Suppl.)     

Purinoceptors on blood platelets: further pharmacological and clinical evidence to suggest the presence of two ADP receptors

Summary. Platelet aggregation by ADP plays a major role in the development and extension of arterial thrombosis. The antithrombotic thienopyridine compounds ticlopidine and clopidogrel have proved useful tools to investigate the mechanisms of ADP-induced platelet activation. In essence, although clopidogrel has been shown to completely and selectively block ADP-induced platelet aggregation, G protein activation and inhibition of adenylyl cyclase, this drug does not affect shape change and Ca²⁺ influx. Binding studies, using the non- hydrolysable ligand [³³P]2MeSADP, have shown that human platelets contain about 600 high-affinity binding sites for 2MeSADP (K_d∼ 5 niw). These sites present pharmacological characteristics of a P_2T receptor. Clopidogrel treatment reduces the number of sites by 70% on rat platelets (from 1200 to 450) and leaves the residual binding sites resistant to clopidogrel. Moreover, patients with congenital impairment of ADP-induced platelet aggregation but normal shape change display very low levels of [³³P]2MeSADP binding sites.The current data thus strongly suggest the presence of two ADP receptors, one responsible for shape change and rapid Ca²⁺ influx and the other a Gi protein-coupled receptor responsible for Ca²⁺ mobilization from internal stores, inhibition of adenylyl cyclase and platelet aggregation.     

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.     

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.