Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian <Emphasis Type="Italic">Dileptis anser</Emphasis>

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca²⁺ was studied and it was found that calcium cations at concentrations of 0.5–10 μM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca²⁺ concentrations to 100 μM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca²⁺. Calmodulin antagonists—chlorpromazine, W-7, and W-5—caused a dose-dependent decrease of the enzyme activity stimulated by 5 μM Ca²⁺ with IC₅₀ values of 35, 137, and 174 M, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 μM Ca²⁺, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 μM calcium ions, but were markedly inhibited by 100 μM Ca²⁺. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca²⁺, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity depends on calcium cations and possibly is regulated by Ca²⁺/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.     

cAMP-dependent regulation of Ca2+ channels expressed inXenopus oocytes

Rat forebrain- and heart-derived mRNA were used to express Ca²⁺ channels inXenopus oocytes to study their cAMP-dependent regulation. Forebrain and heart mRNA-directed Ca²⁺ channel currents (I _Ba, 40 mM Ba²⁺ were used as a charge carrier) showed similar voltage dependence and macroscopic kinetics but different pharmacology, which allowed us to attribute them to N- and L-type, respectively. Brain mRNA-directedI _Ba was insensitive to the dihydropyridine (DHP) antagonist nitrendipine and the agonist Bay K 8644, but could be inhibited by 70% by 1 μM of ω-conotoxin GVIA, whileI _Ba directed by cardiac mRNA was extremely sensitive to DHP. Neither forebrain, nor heart mRNA-directedI _Ba could be augmented by the external applications of the β-agonist isoproterenol (ISO, 10 μM), the adenylate cyclase (AC) activator forskolin (FSK, 10 μM), the phosphodiesterase inhibitor IBMX (200 μM), or their mixtures. “Cardiac”I _Ba was also unresponsive to the external applications of a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (500 μM), as well as to the direct intracellular infusion of cAMP (300 μM). Blockade of cAMP-dependent phosphorylation pathway by intracellular perfusion of the oocytes with 200 μM Rp-cAMP plus 200 μM of a synthetic protein kinase A (PKA) inhibitor peptide also exerted no effect on the basal level ofI _Ba, suggesting that the expressed Ca²⁺ channels are not fully phosphorylated in the resting state. Measurements of the concentration of cAMP in the control and heart mRNA-injected oocytes, using an enzyme-immunoassay system, showed that they display a similar basal cAMP concentration (2.0–2.5 μM); however, application of ISO + FSK increased the cAMP concentration 2- to 3-fold in mRNA-injected oocytes, but not in control oocytes. Thus, our data demonstrate that injection of rat cardiac mRNA intoXenopus oocytes results in the expression of receptor-stimulated AC and L-type Ca²⁺ channels, which do not respond to cAMP or PKA inhibitors. Unresponsiveness to cAMP-dependent regulation is not channel type-specific, since N-type Ca²⁺ channels expressed by means of forebrain mRNA are also insensitive to such regulation. Unresponsiveness of the channels to cAMP-mediated regulation is most probably due to lack/inaccessibility of PKA-dependent phosphorylation site(s), or loss of functional significance of phosphorylation.     

Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypotonic shock indunaliella viridis

An adenylate cyclase activity of 16.02±1.03 pmol cAMP produced min⁻¹ (mg protein)⁻¹ was detected in a cell homogenate ofDunaliella viridis, a unicellular halotolerant green alga. It was present in both the membrane fraction and soluble fraction separated from the homogenate. Adenylate cyclase activity in the homogenate was activated by 1μM GTPγS but not by Ca²⁺+calmodulin, suggesting this enzyme to be regulated by a G-protein. A phosphodiesterase activity of 23.12±15.03 pmol cAMP decomposed min⁻¹ (mg protein)⁻¹ was found in the homogenate. These activities suggest the presence of a cAMP mediated signal transduction system inDunaliella. Cells, transferred from 1.7 M NaCl medium to 1 M NaCl, showed rapid increase in cAMP within 2 min to about 1.5 times the original concentration (from 2.4±0.2 to 3.9±0.2 pmol per 10⁸ cells) which was recovered in 30 min.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca²⁺. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca²⁺. Specifically, 5 µM thapsigargin or 3 µM A-23187 (calcium ionophore), both of which increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca²⁺, suggesting that it is dependent on Ca²⁺ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C₂ (i.e., cells that elevate [Ca²⁺]_i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca²⁺ may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction     

Inhibitory effect of calcium-binding protein regucalcin on Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca²⁺/calmodulin-dependent cyclic nucleotide (AMP) phosphodiesterase activity in rat liver cytosol was investigated. The addition of Ca²⁺ (50 µM) and calmodulin 160 U/ml in the enzyme reaction mixture caused a significant increase in cyclic AMP phosphodiesterase activity. This increase was inhibited by the presence of regucalcin (0.5-3.0 µM); the inhibitory effect was complete at 1.0 µM. Regucalcin (1.0 µM) did not have an appreciable effect on basal activity without Ca²⁺ and calmodulin. The inhibitory effect of regucalcin was still evident even at several fold higher concentrations of calmodulin (160–480 U/ml). However, regucalcin (1.0 µM) did not inhibit Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity in the presence of 100 and 200 µM Ca²⁺ added. Meanwhile, Cd² (25–100 µM)-induced decrease in Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity was not reversed by the presence of regucalcin (1.0 µM). The present results suggest that regucalcin can regulate Ca²⁺/calmodulin-dependent cyclic AMP phosphodiesterase activity due to binding Ca²⁺ in liver cells.     

In Vitro Stimulation of Protein Kinase C by Melatonin

It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca²⁺ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca²⁺ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [³H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 M) and protein kinase C inhibitors (100 M) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [³H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca²⁺. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca²⁺ dependent proteins.     

Alpha-Ketoisocaproic Acid Increases Phosphorylation of Intermediate Filament Proteins from Rat Cerebral Cortex by Mechanisms Involving Ca<Superscript>2+</Superscript> and cAMP

We have previously described that α-ketoisocaproic acid (KIC), the main metabolite accumulating in maple syrup urine disease (MSUD), increased the in vitro phosphorylation of cytoskeletal proteins in cerebral cortex of 17- and 21-day-old rats through NMDA glutamatergic receptors. In the present study we investigated the protein kinases involved in the effects of KIC on the phosphorylating system associated with the cytoskeletal fraction and provided an insight on the mechanisms involved in such effects. Results showed that 1 mM KIC increased the in vitro incorporation of ³²P into intermediate filament (IF) proteins in slices of 21-day-old rats at shorter incubation times (5 min) than previously reported. Furthermore, this effect was prevented by 10 μM KN-93 and 10 μM H-89, indicating that KIC treatment increased Ca²⁺/calmodulin- (PKCaMII) and cAMP- (PKA) dependent protein kinases activities, respectively. Nifedipine (100 μM), a blocker of voltage-dependent calcium channels (VDCC), DL-AP5 (100 μM), a NMDA glutamate receptor antagonist and BAPTA-AM (50 μM), a potent intracellular Ca²⁺ chelator, were also able to prevent KIC-induced increase of in vitro phosphorylation of IF proteins. In addition, KIC treatment was able to significantly increase the intracellular cAMP levels. This data support the view that KIC increased the activity of the second messenger-dependent protein kinases PKCaMII and PKA through intracellular Ca²⁺ levels. Considering that hyperphosphorylation of cytoskeletal proteins is related to neurodegeneration it is presumed that the Ca²⁺-dependent hyperphosphorylation of IF proteins caused by KIC may be involved to the neuropathology of MSUD patients.     

Neurochemical mechanisms responsible for depotentiation of synaptic transmission

It was established in experiments on murine hippocampal slices that low-frequency (1 sec⁻¹, 15 min) stimulation of the Schaffer collaterals applied 45 to 60 min after their high-frequency repetitive stimulation (60 sec⁻¹, 0.5 sec) results, in 2/3 of the slices, in reduction of the amplitude of population EPSP recorded from pyramidal neurons of theCA1 area, almost to its level before high-frequency stimulation. Depotentiation was practically completely prevented by application of a non-competitive blocker of NMDA glutamate receptors (GR), ketamine (100 μM), was weakened by a blocker of voltage-dependent L-type Ca²⁺ channels, nifedipine (10 μM), and remained significant after a competitive blocker of the AMPA/kainate receptors, CNQX (10 μM), had been applied to the slices. Depotentiation was significantly reduced by 10 μM of a calmodulin inhibitor, trifluoroperazine, by an increase in the intracellular cAMP concentration caused by activation of A2-adenosine receptors and D5-dopamine receptors, but was resistant to the action of 50 μM of a protein kinase C (PKC) inhibitor, polymixin B. Nootropic compounds possessing anti-amnestic activity enhanced the depotentiation. It is suggested that depotentiation is due to an increase in the intracellular Ca²⁺ concentration, activation of protein phosphatases, and dephosphorylation of pre- and post-synaptic substrates involved in the expression of long-term post-tetanic potentiation of synaptic transmission, which result from cooperative activation of NMDA GR and metabotropic GR.     

Inhibition of ATP-Induced Ca<Superscript>2+</Superscript> Influx by Corticosterone in Dorsal Root Ganglion Neurons

In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons. In the present study, the effect of corticosterone (CORT) on ATP-induced Ca²⁺ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca²⁺]i). ATP, an algesic agent, caused [Ca²⁺]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced [Ca²⁺]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca²⁺ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated corticosterone had no effect on ATP-induced [Ca²⁺]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced [Ca²⁺]i transients in cultured DRG neurons.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

Expression and regulation of insulin-like growth factor binding protein-1 in the rat uterus throughout estrous cycle

The role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ sequestering of rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Nuclear Ca²⁺-ATPase activity increased linearly in the range of 10–40 M Ca²⁺ addition. With those concentrations, Ca²⁺ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca²⁺-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 M), nicotinamide-adenine dinucleotide (NAD⁺; 2 mM) and zinc sulfate (2.5 and 5.0 M). These reagents caused a significant decrease in the nuclear Ca²⁺ uptake and a corresponding elevation in Ca²⁺ release from the nuclei. Moreover, calmodulin (10 g/ml) increased significantly nuclear Ca²⁺-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 M), an antogonist of calmodulin. The present findings suggest that Ca²⁺-ATPase plays a role in Ca²⁺ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca²⁺-ATPase may evoke Ca²⁺ release from the Ca²⁺-loaded nuclei.     

Suppression of mediator secretion in murine neogenic motor synapses with the participation of L-type Ca<Superscript>2+</Superscript>-channels and ryanodine receptors

L-type Ca²⁺-channel blockers, verapamil (5 μM) and nifedipine (10 μM), have increased the quantum composition of endplate potentials (EPP) and the level of induced rhythmic activity of neogenic synapses. L-type Ca²⁺-channel activator BAY K 8644 (1 μM) has a decreased mediator secretion level. Nifedipine (10 μM) has not changed the frequency and amplitude of diminutive EPPs in the dormant state or during potassium depolarization. Blocking of the prejunctional ryanodine receptor with ryanodine (10 μM) led to an increase in the single EPP quantum composition that was qualitatively similar to nifedipine and verapamil, but more marked, and also caused the reinforcement of mediator release during the rhythmic EPP salvo. Ryanodine receptor activation with ryanodine (1 μM) resulted in reduction of the quantum composition of single and rhythmic EPPs. This effect was partially prevented with nifedipine (10 μM).     

Regulation of Adenylyl Cyclase Signaling System in Cell Cultures of Infusoria Dileptus anser and Tetrahymena pyriformis by Peptides of Insulin Superfamily

Hormone-sensitive adenylyl cyclase signaling system (ACS) provides transduction of a wide spectrum of hormonal signals in cells of the higher eucaryotes. At the same time, ACS in the lower eucaryotes at present is practically not studied. We studied regulatory effects on ACS of the infusoria Dileptus anser and Tetrahymena pyriformis of peptide hormones of the higher eukaryotes—insulin, IGF-1, and relaxin, whose action on ACS of the higher eucaryotes was the subject of our earlier studies. The action of these hormones at concentrations of 10^–10–10^–8 M on the AC activity in infusoria had clearly stimulating character, the dose–effect curves being of a bell-shaped form with a maximum of the stimulating effect of the hormones at concentrations of 10^–9–10^–8 M. the shape of the curves and the value of the stimulating effect of the peptide hormones depended substantially on the level of the AC basal activity in homogenates of infusorian cell cultures. All the hormones (10^–8 M) stimulated GTP-binding activity of G-proteins. It was shown by the example of relaxin that its stimulating effect on GTP-binding in infusorian cells was dose-dependent and increased in the range of hormone concentrations from 10^–10 to 10^–8 M to reach its maximum at concentrations of 10^–8–10^–7 M. In the presence of suramin, an inhibitor of heterotrimeric G-proteins, the stimulating effects of the hormones on the GTP-binding and the AC activity decreased essentially or were absent completely. This indicates that the heterotrimeric G-proteins are ones of components of the signaling cascade that mediates regulatory effects of the hormones of the insulin group on the AC activity in infusorian cell cultures. Based on the obtained data, it is suggested that the basic molecular mechanisms of regulation of ACS by insulin and the related peptides that are similar to those found in the higher vertebrates already begin to be formed as early as at the level of the lower eucaryotes.     

Comparative study of effects of cadmium cations in free and chelated forms on activity of glutathione S-transferase, growth, and endocytosis in culture of the infusoriumTetrahymena pyriformis

Effects of cadmium cations in free (Cd²⁺) and chelated with EDTA (Cd²⁺-EDTA) forms were studied on growth, endocytosis, and activity of glutathione S-transferase (GT) in the free-living infusoriaTetrahymena pyriformis. It is shown that the cytotoxicity of Cd²⁺ in the free form at a concentration of 10 μM is much higher than of the Cd²⁺-EDTA complex at the equimolar concentration. Even at a low concentration (2 μM), Cd²⁺ produces an inhibition of the growth rate and endocytosis in theT. pyriformis culture, while the Cd²⁺-EDTA complex suppresses these functions insignificantly. Cd²⁺ in the free form at concentrations of 10 and 100 μM reduced activity of glutathione S-transferase by 39 and 61%. The chelated Cd²⁺-EDTA complex at these concentrations inhibited the GT activity by 5 and 55%, respectively.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.