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.     

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.     

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.     

Effects of Ethanol on NMDA Receptors in Brain: Possibilities for Mg2+ -Ethanol Interactions

The major excitatory neurotransmitter in the CNS is L-glutamate, and one of the subtypes of L-glutamate receptors, the N -methyl-D-aspartate (NMDA) subtype, has been found to be quite sensitive to inhibition by low concentrations of ethanol (5–50 mm). The NMDA receptor-ion channels are unique in that they exhibit a voltage-dependent blockade by physiological concentrations of Mg²⁺, a blockade that is relieved as the cell membrane is depolarized. Several lines of evidence also suggest that the activity of this receptor-channel complex may be regulated through a high-affinity Mg²⁺ site, which is distinct from the channel-blocking site and could even be located on the extracellular domain of the protein. This high-affinity Mg²⁺ site has been shown to increase the binding of N -[1-(2-thienyl) cyclohexyl]piperidine within the ion channel, as well as the binding of competitive antagonist such as 3-(±)-carboxypiperazine-4-yl)-[1,2]-propyl-1-phosphonic acid and the receptor coactivator glycine. The relationship between the acute effects of ethanol on receptor activation and the regulatory properties of Mg²⁺ is not yet known, although the hypomagnesemia that occurs in chronic alcoholism could certainly have implications for receptor function. A significant amount of molecular characterization of the multiple isoforms of the NMDA receptor-ion channel will be required before the role of Mg²⁺ can be clarified and any relationship between Mg²⁺ regulation and ethanol inhibition established.     

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.     

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.     

Enhanced Mg2+-ATPase activity in ghosts from HS erythrocytes and in normal ghosts stripped of membrane skeletal proteins may reflect enhanced aminophospholipid translocase activity

Hereditary spherocytosis (HS) is a congenital haemolytic anaemia which is characterized by a great variety of structural defects in the red cell's membrane skeleton and/or deficiencies in particular membrane (skeletal) proteins. Enhanced (Mg²⁺)-dependent adenosine triphosphatase (Mg²⁺-ATPase) activities, varying from 115% to 160%, were invariably found in erythrocyte ghosts derived from 13 HS patients. Similarly, an enhancement of Mg²⁺-ATPase activity by 30% is observed in normal red cell ghosts that have been stripped of the greater part of their membrane skeletal proteins by treatment with a low ionic strength buffer. Reassociation of those stripped ghosts with spectrin reduces the enhanced Mg²⁺-ATPase activity to its original level. Since in both cases, HS ghosts and stripped normal ghosts, the stabilizing effects that the membrane skeleton exerts on the maintenance of an endofacial localization of the aminophospholipids are impaired, the enhanced Mg²⁺-ATPase activity is interpreted to reflect an increased activity of the aminophospholipid translocase. The present observations therefore support a role of the membrane skeleton in the stabilization of phospholipid asymmetry in the red cell membrane and consequently in reducing the energy consumption of the translocase.     

Effect of Glutathione on Inositol 1,4,5-Triphosphate-Induced Ca2+ Release in Permeabilized Hepatocytes from Control and Chronic Ethanol-Fed Rats

The effect of oxidized and reduced glutathione on inositol 1,4,5-trisphosphate (InsP3)-induced Ca²⁺ release from endoplasmic reticular Ca²⁺ stores was studied in digitonin-permeabilized hepatocytes from chronically ethanol-fed rats and pair-fed control animals. The fractional Ca²⁺ release induced by a subsaturating concentration of lnsP3 was significantly enhanced in cells from ethanol-fed rats in the absence of a change in maximal lnsP3-releasable Ca²⁺ pool size, and this difference was not affected by preincubation with reduced glutathione. Incubation with oxidized glutathione (1 mM) increased the efficacy of Ca²⁺ release by subsaturating concentrations of lnsP3 in both control preparations and in cells from ethanol-fed rats. The shift in the InsP3 dose-response curve was not significantly different between the two preparations. These findings suggest that the enhanced efficacy of InsP3-induced Ca²⁺ release in hepatocytes from ethanol-fed rats is not caused by the oxidation of protein-bound thiol groups on the lnsP3 receptor.     

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.     

Energetics of the Na+ Pump in the Heart

Movement of ions across the cell membrane is driven by the free energy released from ATP hydrolysis. Here we will briefly review the chemistry of Na⁺, K⁺-ATPase reaction; we will also review some recent results defining the relationship between the energetic driving force and Na⁺ efflux by the Na⁺ pump in the normal myocardium and then discuss this relationship for pathophysiologic states in the heart.     

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).     

Adenosine Triphosphatase Activity of the Red Cell Membrane in Paroxysmal Nocturnal Haemoglobinuria

The activity of the ATPase complex of red cell ghosts as well as the activity of the ATPase components, Mg²⁺ ATPase and Mg²⁺, Na⁺ and K⁺ -dependent ATPase was studied in patients with paroxysmal nocturnal haemoglobinuria (PNH). In ghosts prepared by osmotic haemolysis a significantly higher ATPase activity was found in PNH cells than in normal red cells. Ghosts exposed to the effect of saponin showed a significant increase of ATPase activity both in normal and PNH red cells; however, the increase of activity was markedly higher in PNH ghosts than in ghosts of normal red cells. Both Mg²⁺-dependent and Mg²⁺, Na⁺ and K⁺-dependent ATPase contribute to this increase. The protein content in osmotic ghosts of PNH red cells was higher than normal; but after treating these ghosts with saponin, no marked difference in protein content was found.
By comparison with the results of ATPase activity obtained using red cells from non-splenectomized individuals with hereditary spherocytosis with similar reticulocyte values it is assumed that the increase of ATPase activity is not due to younger cells in the blood but is a further manifestation of the abnormality of the cell membrane in PNH. ATPase activity is present mostly in the inactive form in the red cell membrane and is activated (unmasked) by structural damage of the cell membrane. It is suggested that the increased ATPase activity in the PNH red cell membrane represents a manifestation of abnormal structural arrangement which is more susceptible to damage.
The deviations found in the content and metabolism of organic phosphates are probably caused by the younger population of red cells in PNH.     

Membrane Time Constant During Internal Defibrillation Strength Shocks in Intact Heart: Effects of Na+ and Ca2+ Channel Blockers

Introduction: We assessed defibrillation strength shock-induced changes of the membrane time constant (τ) and membrane potential (ΔVm) in intact rabbit hearts after administration of lidocaine, a sodium (Na⁺) channel blocker, or nifedipine, a L-type calcium (Ca²⁺) channel blocker.
Methods and Results: We optically mapped anterior, epicardial, electrical activity during monophasic shocks (±100, ±130, ±160, ±190, and ±220 V; 150 μF; 8 ms) applied at 25%, 50%, and 75% of the action potential duration via a shock lead system in Langendorff-perfused hearts. The protocol was run twice for each heart under control and after lidocaine (15 μM, n = 6) or nifedipine (2μM, n = 6) addition. τ in the virtual electrode area away from the shock lead was approximated with single-exponential fits from a total of 121,125 recordings. The same data set was used to calculate ΔVm. We found (1) Under all conditions, there is inverse relationship between τ and ΔVm with respect to changes of shock strength, regardless of shock polarity and phase of application: a stronger shock resulted in a larger ΔVm, which corresponded to a smaller τ (faster cellular response); (2) Lidocaine did not cause appreciable changes in either τ or ΔVm versus control, and (3) Nifedipine significantly increased both τ and ΔVm in the virtual cathode area; in contrast, in the virtual anode area, this effect depended on the phase of shock application.
Conclusion: τ and ΔVm are inversely related. Na⁺ channel blocker has minimal impact on either τ or ΔVm. Ca²⁺ blocker caused polarity and phase-dependent significant changes in τ and ΔVm.     

Eosinophilia associated with proliferation of CD3+4−8−αβ+ T cells with chromosome 16 anomalies

We describe a patient with eosinophilia and an abnormal CD3⁺4⁻8⁻αβ⁺ T-cell population. Chromosomal analysis of sorted CD3⁺4⁻8⁻ cells revealed abnormal karyotypes on chromosome 16. In the presence of IL-2 the production of IL-5 from CD3⁺4⁻8⁻ cells was higher than that from CD3⁺4⁺/8⁺ cells. Eosinophil survival-enhancing activity in the patient serum was inhibited by a combination of anti-IL-5 and anti-GM-CSF monoclonal antibodies. These data suggest that increased production of IL-5 and GM-CSF from the abnormal CD3⁺4⁻8⁻ cells might cause eosinophilia.     

Na+/H+ exchanger mediates TNF-α-induced hepatocyte apoptosis via the calpain-dependent degradation of Bcl-xL

Background and Aim:  It is well known that tumor necrosis factor-α (TNF-α) induces hepatocyte apoptosis and contributes to liver diseases. However, the exact mechanisms are not well understood.
Methods:  In the present study, we reported that Na⁺/H⁺ exchanger (NHE) is involved in TNF-α-induced hepatocyte apoptosis.
Results:  TNF-α time dependently induced an increase in NHE activity in hepatocytes, but cariporide, an NHE inhibitor, blocked the TNF-α-induced increase of NHE activity in a dose-dependent manner. Increased NHE activity induced by TNF-α was associated with increased intracellular calcium (Ca²⁺_i) concentration and calpain activity. Cariporide reversed these effects induced by TNF-α. In addition, TNF-α downregulated Bcl-xL, an anti-apoptotic protein, but not mRNA levels. The inhibition of either calpain or NHE blocked the TNF-α-induced decrease of the Bcl-xL protein. TNF-α did not change the pro-apoptotic Bax and Bak protein levels. Cariporide, calcium remover 1,2-bis (2-aminophenoxy) ethane-N,N,N0,N0–tetraacetic acid, or calpain inhibitor benzyloxycarbonyl-leucyl-leucinal attenuated TNF-α-induced hepatocyte apoptosis.
Conclusion:  TNF-α via NHE results in hepatocyte apoptosis through the calcium/calpain/Bcl-xL pathway.     

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.     

Establishment of an IL-2-dependent cell line derived from 'nasal-type' NK/T-cell lymphoma of CD2+, sCD3−, CD3ɛ+, CD56+ phenotype and associated with the Epstein-Barr virus

A novel interleukin-2 (IL-2)-dependent cell line, HANK1, was established from a patient with CD56⁺ NK/T-cell lymphoma arising in the retroperitoneum. Morphologically, HANK1 is a pleomorphic large cell line with irregular nuclei, which contains azurophilic granules in the cytoplasm. Immunophenotypic analysis showed that HANK1 expressed CD2, CD3ɛ, CD56, TIA-1, granzyme B, and HLA-DR, but no other T-lineage markers. These features were the same as seen in the original tumour, and are highly characteristic of nasal and 'nasal-type' NK/T-cell lymphoma as described in the proposed W.H.O. classification. Genotypically, this cell line also demonstrated the germline configuration of the T-cell receptor β, γ and the immunoglobulin heavy chain genes and clonal integration of the Epstein-Barr virus (EBV) together with antigen expression with a type II latency pattern (LMP-1⁺ and EBNA2⁻). Furthermore, Southern blot analysis using the EBV termini as probes confirmed its derivation from the original lymphoma, and revealed that it contained multiple copies of the EBV genome. Dose-dependent growth on IL-2 was observed in an in vitro study with a doubling time of 3 d at maximal stimulation. These data indicate that HANK1 seemed to preserve the biological characteristics of the original tumour and therefore may serve as a good model for the further analysis of unusual 'nasal-type' NK/T-cell lymphoma.     

Blastogenic responses, interleukin-2 production and interleukin-2 receptor expression on CD4+ and CD8+ lymphocytes in rhesus monkeys experimentally inoculated with Loa loa

To better understand cellular responses in loiasis infection, in vitro blastogenesis of peripheral blood mononuclear cells (PBMC) to filarial antigen was assessed in 12 Loa loa -inoculated rhesus monkeys over a two-year period. Cellular reactivity to antigen was observed between 10–35 weeks postinoculation (WPI), but had declined by week 50. The roles of interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression on CD4⁺ and CD8⁺ T cells in regulating the response to antigen were examined during the initial (57 WPI) and late (92 WPI) time points of the observed diminished reactivity to antigen. The levels of IL-2 in antigen cultures at both time points were not significantly different from those in unstimulated cultures. Also, exogenous IL-2 partially reversed the PBMC response to antigen. The percentages of CD4⁺ and CD8⁺ T cells expressing IL-2R in antigen cultures at 57 WPI were not different from those of control animals. Likewise at 92 WPI, the percentage of CD4⁺ T cells expressing IL-2R in antigen cultures, were not increased above those of control animals. In contrast, the percentage of CD8⁺ T cells expressing IL-2R in antigen cultures were significantly increased above those of control animals ( P  < 0.0001), coinciding with an increase in CD8⁺ T cell numbers in these cultures. The data show that factors besides IL-2, and probably an imbalance in the percentages of CD4⁺ and CD8⁺ T cells bearing IL-2R in antigen cultures, may contribute to the diminished reactivity to antigen in L. loa -inoculated rhesus monkeys .     

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.     

Effect of Chronic Ethanol Treatment on Ca2+-Inhibited Adenylyl Cyclase in Mouse Striatum

In the present study, to investigate the possibility that chronic ethanol treatment might alter Ca²⁺-inhibited type 5 adenylyl cyclase (AC) activity, we examined the effect of chronic ethanol treatment on striatal dopaminergic signal transduction, especially the AC system, in mice. We fed male C57BL/6 mice for 7 days with a 5% ethanol-containing or control liquid diet. Basal and forskolin-stimulated AC activities were reduced in striatal membranes of ethanol-treated mice. 5'-guanylylimidodiphosphate-stimulated AC activity was also decreased in ethanol-treated mice. But no significant differences were observed in the levels of the guanine nucleotide binding protein subunits Gsα and Gi_1&2α determined by immunoblotting, between ethanol-treated and control mice. These results indicated that the function of the catalytic subunit of AC was decreased in the straitum of chronically ethanol-treated mice. We further examined the inhibitory regulation of AC activity in the context of a change of type 5 AC. Inhibition of forskolin-stimulated AC activity by 10 μM free Ca²⁺ was smaller in ethanol-treated mice than in control mice. However, the protein level of type 5 AC in the striatum, determined by immunoblotting, was not significantly different between ethanol-treated and control mice. These findings suggest that Ca²⁺-inhibited, presumably type 5, AC activity is reduced in mouse striatum by chronic ethanol treatment, and that this reduction is not due to a decrease in type 5 AC expression.