Absence of 1-Methyladenine Production in Follicle Cells Obtained from Starfish Ovaries in the Post-Spawning Season

Follicle cells from the ovaries of starfish Asterina pectinifera collected in the breeding season and then kept in an aquarium for three months did not produce 1-methyladenine (1-MeAde) in response to genad-stimulating substance (GSS). The cyclic AMP content of follicle cells of the ovaries was much lower in the post-spawning season than in the spawning season. In the post-spawning season, the cyclic AMP level was increased slightly by GSS, but nor insufficiently for production of 1-MeAde. Addition of 3-isobutyl-1-methylxanthine, a potent phosphodiesterase inhibitor, stimulated the productions of GSS-induced 1-MeAde and cyclic AMP. Adenylate cyclase in membrane preparations of follicle cells from ovaries in the post-spawning season was stimulated by nonhydrolyzable GTP analogs and forskolin. An experiment on ADP-ribosylation with [α-³²P]NAD in the presence of cholera toxin and pertussis toxin showed the presence of two types (stimulatory and inhibitory) of guanine nucleotide-binding regulatory proteins (G-proteins). However, GSS has no effect on the adenylate cyclase activity regardless of the presence of GTP. These findings suggest that GSS does not bind to its receptor in follicle cells of the overy in the post-spawning season, although these cells possess G-proteins and adenylate cyclase. Thus the absence of 1-MeAde production by follicle cells obained from ovaries in the post-spawning season appears to be due to lack of receptor protein for GSS.     

Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi

Sperm-activating and -attracting factor (SAAF) released from the ascidian, Ciona savignyi , was partially purified from egg seawater with ethanol extraction and separation with the two-phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium-free seawater (CaFSW), but activated the both in the presence of Ca²⁺. Sperm activation by SAAF in Ca²⁺-containing medium was inhibited by flunarizine, a T-type Ca²⁺channel antagonist, but L-type Ca²⁺channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline-activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca²⁺they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca²⁺influx through T-type Ca²⁺channel, and that Ca²⁺alone mediates the sperm chemotaxis in Ciona .     

Tyrosine Hydroxylase in "Leaky" Adrenal Medullary Cells: Evidence for In Situ Phosphorylation by Separate Ca2+ and Cyclic AMP-Dependent Systems

Abstract: The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca²⁺, the other on cyclic AMP. The Ca²⁺-dependent system is half-maximally activated by 1-2 μ M Ca²⁺ and 0.5 m M ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 m M ) does not inhibit significantly Ca²⁺-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 μ M cyclic AMP and about 0.3 m M ATP. Ca²⁺-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10⁻⁸ M Ca²⁺ plus 2 × 10⁻⁵ M cyclic AMP or of 10⁻⁵ M Ca²⁺, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca²⁺-dependent system, whereas the other site(s) are phosphorylated by both the Ca²⁺- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.     

Change in Intracellular Calcium Ions upon Maturation in Starfish Oocytes

A transient increase in intracellular Ca²⁺ upon maturation in starfish oocyte was revealed by light emission of aequorin microinjected into the cell. One minute application of 1-methyladenine (1-MeAde) to a limited area of the oocyte surface was sufficient to induce the Ca²⁺ transient over the entire cell though it did not induce the germinal vesicle breakdown (GVBD). Ten minutes application of 1-MeAde induced a similar Ca²⁺ transient followed by GVBD. Even when the transient increase of Ca²⁺ was inhibited by injecting EGTA into the oocyte, 1-MeAde treatment for a long period induced GVBD. These facts indicate that the Ca²⁺ increase is neither necessary nor sufficient for maturation of the starfish oocyte.
When the oocyte, which had been treated with 1-MeAde for 1 min at a limited area around the animal pole, was treated again with 1-MeAde for 10 min starting about 15 min after the first treatment, a Ca²⁺ transient similar to the first one was induced and was followed by GVBD. By contrast, in the oocyte treated with 1-MeAde at an area around the vegetal pole, neither Ca²⁺ transient nor GVBD was induced by the second treatment with 1-MeAde. These results indicate a difference in responsiveness to the hormone between the animal hemisphere and the vegetal hemisphere of the oocyte.     

Involvement of cyclic adenosine 3′,5′-monophosphate in methylation during 1-methyladenine production by starfish ovarian follicle cells

Summary

Resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeAde) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). With respect to 1-MeAde production by follicle cells of the starfish, Asterina pectinifera, (1) the action of GSS is initiated by a receptor mediated activation of G-proteins, resulting in the activation of adenylate cyclase and cyclic AMP (cAMP) formation; (2) 1-MeAde produced under the influence of GSS is not prestored within the follicle cells but is newly synthesized from a 1-MeAde precursor; (3) AMP plays an important role in the process of methylation during 1-MeAde biosynthesis induced by GSS.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

Calcium Potentiates Cyclic AMP Stimulation of Pineal Arylalkylamine N-Acetyltransferase

Abstract: Pineal arylalkylamine N -acetyltransferase ( N -acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca²⁺ concentration ([Ca²⁺]_i), and it is not known whether Ca²⁺ is involved in regulating N -acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca²⁺ enhances the effects of cyclic AMP on N -acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N ⁶, 2'- O -dibutyryladenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphothioate, Sp-diastereomer) were examined in combination with [Ca²⁺]_i protagonists (A23187, ionomycin, and phenylephrine). All [Ca²⁺]_i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8-bromoadenosine 3',5'-cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca²⁺ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N -acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.     

Melatonin Biosynthesis in Cultured Chick Retinal Photoreceptor Cells: Calcium and Cyclic AMP Protect Serotonin N-Acetyltransferase from Inactivation in Cycloheximide-Treated Cells

Abstract: The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N -acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 µ M forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of ∼30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K⁺, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K⁺ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca²⁺ influx through dihydropyridine-sensitive channels. MDL-12330A, an inhibitor of K⁺-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K⁺. This result suggests that the effect of Ca²⁺ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization-evoked Ca²⁺ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Nature of the 1-Methyladenine-Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1-methyladenine (1-MeAde) in the surrounding medium for a definite period. The length of the 'hormone-dependent phase' (HDP) in Asterina pectinifera , which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1-MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation-inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1-MeAde and DTT. Isotonic CaCl₂, the Ca²⁺ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca²⁺, had additive effects with 1-MeAde. These results suggest that part of the action of 1-MeAde is to release Ca²⁺ in the oocyte cytoplasm.     

Cross Talk Among Cyclic AMP, Cyclic GMP, and Ca2+ -Dependent Intracellular Signalling Mechanisms in Brain Capillary Endothelial Cells

Abstract: C-type natriuretic peptide and sodium nitroprusside, a nitric oxide donor molecule, induced large increases in cyclic GMP formation in cultured rat brain capillary endothelial cells. Isoproterenol, a potent agonist of adenylate cyclase, potentiated the actions of C-type natriuretic peptide and of sodium nitroprusside. These actions were not observed in the presence of isobutylmethylxanthine and were mimicked by forskolin. Endothelin-1 had no action on basal cyclic GMP levels. It reduced cyclic GMP formation induced by C-type natriuretic peptide and sodium nitroprusside by about 50%. These actions involved an ET_A receptor subtype and a Ca²⁺-dependent and protein kinase C-independent mechanism. Finally, increasing cyclic GMP slightly prolonged intracellular Ca²⁺ transients induced by endothelin-1. The results suggest the presence of extensive cross talk among cyclic AMP, cyclic GMP, and Ca²⁺-dependent mechanisms in endothelial cells of brain microvessels. The relevance of the results to the regulation of the blood-brain barrier permeability is discussed.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Coupling of Corticotropin-Releasing Hormone Receptors to Adenylyl Cyclase in Human Y-79 Retinoblastoma Cells

Abstract: In human Y-79 retinoblastoma cells, corticotropin-releasing hormone (CRH) stimulates adenylyl cyclase activity and increases cyclic AMP accumulation. Different CRH analogues mimic the CRH stimulation of adenylyl cyclase and show similar sensitivity to the CRH receptor antagonist α-helical CRH_9–41. Vasoactive intestinal peptide (VIP) also increases the enzyme activity but less potently than CRH, and its effect is counteracted by the VIP receptor antagonist [ d - p -Cl-Phe⁶,Leu¹⁷]VIP. The VIP antagonist does not affect the response to CRH. The CRH-stimulated adenylyl cyclase activity is amplified by Mg²⁺, is inhibited by submicromolar concentrations of Ca²⁺, and requires GTP. Moreover, the CRH stimulation is reduced by pretreatment of cells with cholera toxin and by incubation of membranes with the RM/1 antibody, which recognizes the C-terminus of the α subunit of G_s. In immunoblots, the RM/1 antibody identifies a doublet of 45 and 52 kDa. Two proteins of similar molecular weights are ADP-ribosylated by cholera toxin. These data demonstrate that in human Y-79 retinoblastoma cells, specific CRH receptors stimulate cyclic AMP formation by interacting with G_s and by affecting a Ca²⁺-inhibitable form of adenylyl cyclase.     

A Charybdotoxin-Sensitive, Ca2+-Activated K+ Channel with Inward Rectifying Properties in Brain Microvascular Endothelial Cells: Properties and Activation by Endothelins

Abstract: A charybdotoxin-sensitive, Ca²⁺-activated K⁺ channel was identified in cultured rat brain capillary endothelial cells by using conventional single-channel recording techniques and ⁸⁶Rb⁺-influx and efflux experiments. Channel activity was dependent on the presence of Ca²⁺ on the cytosolic face of the membrane with a threshold concentration of 100 n M . It was inhibited by charybdotoxin (IC₅₀ 30 n M ) and quinine (IC₅₀ 0.1 m M ) but not by apamin. K(Ca) channels showed unusual inward rectifying properties under asymmetrical ionic conditions. They were activated by endothelin-1 (EC₅₀ 0.7 n M ) and endothelin-3 (EC₅₀ 7–10 n M ). The actions of endothelins were prevented by BQ-123 ( K _i = 8 n M ) in a competitive fashion, hence suggesting the involvement of an ET_A-receptor subtype. The channel activity was unaffected by cyclic AMP- or cyclic GMP-elevating agents. The possible role of the intermediate conductance, Ca²⁺-activated K⁺ channels for mediating K⁺ movements across the blood-brain barrier is discussed.     

Transient Increase of Cyclic AMP Induced by Glutamate in Cultured Neurons from Rat Spinal Cord

Abstract: We demonstrated that glutamate increased the cyclic AMP level in cultured neurons from rat spinal cord. A bath application of glutamate (300 µ M ) elicited a rapid increase of the cyclic AMP concentration reaching a level three times as high as the basal level in ∼3 min, and its content then decreased to the control level in 15 min. The increase was not observed in a Ca²⁺-free medium and was inhibited by an antagonist of NMDA receptors or a voltage-sensitive Ca²⁺ channel blocker. Preincubation with W7 also inhibited the glutamate-evoked cyclic AMP increase. NMDA, aspartate, and high-K⁺ conditions also induced a cyclic AMP increase; however, a decreasing phase did not follow. The decreasing phase was observed when (2 S ,1' S ,2' S )-2-(carboxycyclopropyl)-glycine, a potent agonist for metabotropic glutamate receptors, was combined with NMDA. These results suggest that the cyclic AMP increase is mediated by a Ca²⁺ influx via both NMDA receptors and voltage-sensitive Ca²⁺ channels followed by an activation of the Ca²⁺/calmodulin system, and the decreasing phase observed in the case of glutamate exposure is due to the activation of the metabotropic glutamate receptors.     

Arginine-Modulated Receptor-Activated Calcium Influx via a NO/Cyclic GMP Pathway in Human SK-N-SH Neuroblastoma Cells

Abstract: The effects of arginine on calcium mobilization in human SK-N-SH neuroblastoma cells were examined. It was found that arginine potentiated an increase in carbachol-induced Ca²⁺ from the external Ca²⁺ influx as opposed to an internal Ca²⁺ release from intracellular pools. The potentiation effect of arginine on carbachol-induced calcium mobilization was mimicked by either 8-bromo cyclic GMP or sodium nitroprusside. In addition, it was found that arginine induced NO production and an increase in cyclic GMP. Moreover, arginine-induced potentiation, NO production, and cyclic GMP increases were all suppressed after the preincubation of cells with N -methyl- l -arginine or N -nitro- l -arginine, nitric oxide synthase inhibitor. It is suggested that the NO production and subsequent cyclic GMP elevation induced by arginine are responsible for the potentiation of carbachol-induced Ca²⁺ increase. Our results show the existence of a NO/cyclic GMP pathway and an interconnection of NO and Ca²⁺ signaling pathways in human SK-N-SH neuroblastoma cells. We also observed that NO, which is produced by endothelial CPAE cells, has a modulating effect on cyclic GMP elevation in human SK-N-SH neuroblastoma cells. The intercellular communication role of NO and its cell-diffusing character may also affect the regulation of nonneuronal cells in their interactions with neuronal cells.     

1-Methyladenine production from ATP by starfish ovarian follicle cells.

1-Methyladenine (1-MeAde), the oocyte maturation-inducing substance in starfish, is produced by ovarian follicle cells upon stimulation with a gonad-stimulating substance (GSS) released from the radial nerves. We have shown previously that GSS causes a reduction in the intracellular levels of ATP coincident with 1-MeAde production. The present study examined whether the adenine molecule of 1-MeAde is directly derived from ATP. When isolated follicle cells from the starfish Asterina pectinifera were preloaded with [U-14C]adenine or [U-14C]adenosine, there was an increase in the intracellular levels of radiolabeled adenine nucleotides, particularly ATP. Following further incubation with GSS, the intracellular levels of radiolabeled ATP decreased, concomitant with a marked increase in the levels of [14C]1-MeAde in the medium. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeAde produced. However, there was no change in the levels of ADP and AMP regardless of the presence or absence of GSS. These findings strongly suggest that 1-MeAde is synthesized from ATP as a substrate in follicle cells under the influence of GSS. Furthermore, using [methyl-3H]methionine, the methyl group of 1-MeAde was found to be derived from methionine. Thus GSS appears to stimulate the synthesis of 1-MeAde from ATP via the methylation process in starfish ovarian follicle cells.     

Effects of Tetanus Toxin on Catecholamine Release from Intact and Digitonin-Permeabilized Chromaffin Cells

Abstract: Tetanus exotoxin inhibited Ca²⁺-dependent cate-cholamine secretion in a dose-dependent manner in digito-nin-permeabilized chromaffin cells. The inhibition was specific for tetanus exotoxin and the B fragment of tetanus toxin; the C fragment had no effect. Inhibition required the introduction of toxin into the cell, and was not seen when intact cells were preincubated with the toxin or toxin fragments. The degree of inhibition was related to the length of preincubation with toxin, as well as the concentration of toxin used. A short preincubation with toxin was sufficient to inhibit secretion, and the continued presence of toxin in the incubation medium was not required during the incubation with Ca²⁺. The inhibition of secretion by tetanus toxin or the B fragment was not overcome with increasing Ca²⁺ concentrations. Tetanus toxin also inhibited catechol-amine secretion enhanced by phorbol ester-induced activation of protein kinase C. Thus, the toxin or a proteolytic fragment of the toxin can enter digitonin-permeabilized cells to interact with a component of the Ca²⁺-dependent exocytotic pathway to inhibit secretion.