On the role of serotonin and 5-methoxy-tryptamine in the regulation of cell division in sea urchin eggs

The presence of serotonin in sea urchin eggs has been ascertained by high-performance liquid chromatography and thin-layer chromatography analysis of tissue-free bioamines. The results show the presence of both serotonin and 5-methoxytryptamine. The role of these substances in the cell division process has been studied by using the serotonin antagonists, gramine and metergoline. Both antagonists cause a significant delay of the cell division which, however, can be prevented by the addition of either 5-hydroxytryptophane, serotonin, or 5-methoxytryptamine. The effect of gramine on the different stages of the cell division process has also been investigated. Neither S phase nor mitosis are affected by the serotonin antagonist, while cleavage is delayed. The effect of serotonin seems mediated by calcium ions and cAMP. Gramine causes a marked increase in radio-calcium efflux from the fertilized egg, and at the same time lowers the cAMP level.     

Serotonin, bradykinin and endothelin signalling in a sheep choroid plexus cell line

The secretion of cerebrospinal fluid by the epithelial cells of choroid plexus is regulated by membrane receptors coupled to adenylyl cyclases or to phospholipase C. These intracellular signalling pathways as their interactions were investigated in a sheep choroid plexus cell line. Endothelin-1, bradykinin and serotonin induced a transient dose-dependent increase in intracellular calcium. EC 50 were 10(-8) M for endothelin-1, 10(-8) M for bradykinin and 10(-6) M for serotonin. Maximal increase in intracellular calcium was comparable for bradykinin and serotonin, but was 3 to 5 fold larger for endothelin-1. Successive stimulations with endothelin-1, serotonin or bradykinin elicited calcium increases similar to single stimulations reflecting absence of heterologous desensitization between these receptors. Forskolin-induced cAMP accumulation was potentiated by bradykinin, but not by serotonin and endothelin-1. This potentiation resulted from an increase in cAMP production rather than to an inhibition of cAMP hydrolysis. These data suggest that serotonin, endothelin-1 and bradykinin each use specific signalling pathways in the sheep choroid plexus cells.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.     

Changes in rodent thyroid hormones and cyclic-AMP following treatment with pineal indolic compounds.

Treatment of rats with pineal indolic compounds 5-methoxytryptophol, 5-hydroxytryptophol and serotonin brought about a significant increase in serum thyroxine levels, while serotonin and melatonin caused an increase in thyroid cAMP content with corresponding decrease in the gland's hormones. The total quantity of cAMP in the thyroid was also increased by melatonin in the organ culture system. All these findings would indicate that some of the pineal indoleamines elicit a direct action on the thyroid by stimulating the adenyl cyclase activity and intrathyroidal cAMP, bringing about increased release of thyroxine into the blood stream, and that this is usually not accompanied by adequate synthesis in the gland. Our observation that continuous darkness, which stimulates pineal activity, also brought about an increase in cAMP, concours with our finding of a stimulatory effect of the indolic compounds on thyroid hormone release.     

The role of cAMP in generating excitatory serotonin responses of neurons in the edible snail

The neurons of the dorsal surface of snail Helix subesophageal ganglia respond similarly to the application of serotonin and the intracellular cAMP injection. These responses represent membrane depolarization. They increase in amplitude with membrane hyperpolarization and have a reverse potential between +10 and -30 mV. Presumably, these responses are associated with increased conductance for several ions. The values of the reverse potentials of serotonin and cAMP responses coincide in 7 out of 17 cells. Phosphodiesterase inhibitor theophylline caused a reversible increase in the amplitude and duration of both serotonin and cAMP responses and, used at a concentration of 1 mM, simulated them. The results obtained meet 2 out of 4 criteria demonstrating that cyclic nucleotides mediate a neurotransmitter response. It is suggested that cAMP may act as a second messenger in excitatory serotonin responses of snail Helix neurons.     

Effect of serotonin and calcium on the supercontractile muscles of the adult blowfly crop

Bioassays and electrophysiological recordings were conducted to determine the role of serotonin and calcium on the supercontractile pump muscles of the diverticulated crop of adult blowflies. Using in situ crop preparations, serotonin was found to significantly increase the rates of contractions of a specific pump in the crop wall, pump P4. The addition of the serotonin antagonist, mianserin, or calcium free saline, both significantly reduced the contraction rates of this pump. Recordings, using suction electrodes from pump P4, confirm the in situ bioassay data and show that serotonin promotes muscle activity in empty crops in which no pump activity is normally observed. Moreover, our data indicate the crucial role of extracellular calcium ions in crop pump contractile activity. These results provide new information on how the crop of adult dipterans is modulated and suggest that serotonin, possibly supplied by neurons in the thoracico-abdominal neural plexus, may be involved in modulating the pumping of crop contents into the midgut for digestion or triggering antiperistalsis from the foregut in the process known as regurgitation or 'bubbling'.     

Calcium regulates the regeneration of cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.     

Calcium Regulates the Regeneration of Cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.     

The role of electro-mechanical and reaction-diffusion systems in the intra-neuron processing of information: effect of in-flow of calcium and intracellular calcium on cAMP activity

Influx of calcium ions cannot control a generatory potential induced by the intraneuronal system because calcium ions enter the cell during impulses. These impulses are the result of problem solving and must not influence directly the generatory potential. Therefore cAMP and not calcium controls the permeability of sodium and potassium channels from the inside of the neuron. However the calcium ions and membrane potential of mitochondria affect the impact of cAMP injections. An increase in the intracellular concentration of free Ca2+ induced by the injection of Ca-EGTA buffer with 5.10(-7) M free Ca2+, electric excitation, uncouplers of oxidative phosphorylation or arsenate leads to an increase of cAMP-dependent depolarization and the inward current. The injection of Ca-EGTA buffer with 10(-5) M free Ca2+ and drop in [Ca2+]in by EGTA as well as generation of impulses after cAMP injection decrease the cAMP effect. As rise in [Ca2+]in activates phosphodiesterase and uncouples oxidative phosphorylation, and vanadate in contrast to arsenate suppresses the cAMP effect, a hypothesis is advanced that activating effect of calcium on cAMP action is associated with neuron deenergization.     

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.     

Functional characteristics of calcium-sensitive adenylyl cyclase of the infusorian <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

The calcium-sensitive forms of adenylyl cyclases (AC) have been revealed in the majority of vertebrate and invertebrate animals, as well as in several representatives of unicellular organisms, including infusoria. We have found for the first time that the AC activity in the infusorian Tetrahymena pyriformis changes in the presence of calcium ions. Calcium ions at concentrations of 0.2–20 μM stimulated the activity of this enzyme, with the maximum of the stimulatory effect being observed at 2 μM Ca²⁺. At a concentration of 100 μM and higher, the calcium cations inhibited the AC activity. Antagonists of calmodulin W-5 and W-7 at concentrations of 20–100 μM decreased the stimulatory effect of 5 μM Ca²⁺, while at the higher concentrations inhibited it completely. Another calmodulin antagonist, chloropromazine, decreased the Ca²⁺-stimulated AC activity only at concentrations of 200–1000 μM. The stimulatory effect of serotonin, EGF, and cAMP on AC activity was enhanced in the presence of 5 μM Ca²⁺. The stimulatory effect of EGF, cAMP, and insulin on AC was decreased in the presence of 100 μM Ca²⁺, while the effect of cAMP was also observed in the presence of calmodulin antagonists (500 μM). At the same time, stimulatory effect of D-glucose did not change in the presence of Ca²⁺ and calmodulin antagonists. The obtained data indicate that, in the infusorian T. pyriformis, there are calcium-sensitive forms of AC that can be stimulated by EGF, cAMP, insulin, and serotonin.     

Regulation of the cell cycle of B-lymphocytes in mice by substances elevating the levels of intracellular cAMP and cGMP

Spontaneous velocity sedimentation of B lymphocytes activated by intraperitoneal injection of ovalbumin into mice was used to obtain cell cycle synchronized cells, evidenced by differences in the incorporation of labeled precursors of protein and nucleic synthesis (14C-methionine and 3H-thymidine). The effects of acetylcholine and adrenaline, cAMP and cGMP on the intensity of 3H-thymidine incorporation into mouse B lymphocytes and on the amount of the cells entering mitosis were examined. It was shown that acetylcholine is capable of stimulating whereas adrenaline of inhibitin B lymphocyte entry into the stage of DNA synthesis and egress of these cells from the stage of DNA synthesis to the stage of mitosis. Adrenaline was found to have a reciprocal action. The acetylcholine effect could be mimetized by exogenous cGMP, that of adrenaline by cAMP. Stimulation of the G1/S transition was mediated by intracellular calcium ions but did not depend on exocellular calcium.     

Cyclic AMP, 5-HT, and the modulation of transmitter release at the crayfish neuromuscular junction

In this study it was found that several agents which elevate cAMP levels in cells also increase dramatically the quantity of transmitter released from crayfish excitatory nerve terminals in response to a stimulus. With respect to time course and magnitude, the increase produced by one of these agents, the cyclic nucleotide phosphodiesterase inhibitor Squibb 20,009 (SQ 20,009), is unlike any reported for such a drug at a synapse. Additionally, SQ 20,009 potentiated the facilitation of transmitter release produced by serotonin (5-HT) at this synapse. These results establish a possible role for cAMP in the control and modulation of transmitter release at the crayfish neuromuscular junction (NMJ). They further suggest that 5-HT functions here by activation of a presynaptically located adenylate cyclase.     

Calcium Independence of Phosphoinositide Hydrolysis-Induced Increase in Cyclic AMP Accumulation in SK-N-SH Human Neuroblastoma Cells

Previous work has shown that stimulation of muscarinic receptors in various cell lines increases intracellular cyclic AMP (cAMP) levels. This unusual response has been hypothesized to be mediated by stimulation of calcium/calmodulin-sensitive adenylate cyclase, secondary to inositol trisphosphate (IP3)-mediated calcium mobilization. To test this hypothesis, we stimulated muscarinic receptors in SK-N-SH human neuroblastoma cells while blocking the IP3-mediated rise in intracellular calcium concentration using two different methods. Loading cells with the intracellular calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the carbachol-mediated intracellular calcium release without abolishing the carbachol-mediated increase in cAMP level. Similarly, in cells preexposed to carbachol, the agonist-induced change in intracellular calcium level was blocked, but the cAMP response was not. Thus, both of these methods failed to block the muscarinic receptor-mediated increase in cAMP level, thereby demonstrating that this cAMP level increase is not mediated by a detectable rise in intracellular calcium concentration.     

A study of the change in conductivity of the neuronal membrane caused by a generator potential using a method of computer modeling

Injection of cAMP induces in snail neurons generator potential, which is related to an increase of sodium and decrease of potassium permeability of the neuron outer membrane. A model is proposed which takes into account cAMP diffusion inside the neuron from the injection place and interaction of these molecules with the intercellular system controlling permeability of the outer membrane. Resulting impulse generation induces calcium ions current through the outer membrane. The model also considers calcium diffusion toward cAMP and its effect on the rate of the enzyme work destroying cAMP. Agreement between the calculations of ionic current I(t) and the experiment permits determination of the model parameters and calculation of the observed change of time distribution of nerve impulses when calcium input is significant.     

Different intracellular signals coupled to the antiproliferative action of aqueous crude extract from Larrea divaricata Cav. and nor-dihydroguaiaretic acid on a lymphoma cell line.

In this paper, we report the effect of standard NDGA, as compared to that of an aqueous extract of Larrea divaricata Cav., on BW 5147 lymphoma cell-line proliferation. To determine the mechanism of action, the effects of both on the level of intracellular cAMP, protein kinase C activity and calcium influx were studied. Moreover, the NDGA present in the aqueous extract of the plant was quantified. The aqueous extract and the standard NDGA showed antiproliferative action against these cells. While the antiproliferative activity of the aqueous extract was mediated by an increase in cAMP levels, and inhibition of PKC and calcium influx, the antiproliferative activity of NDGA was related only to the inhibition of PKC. Considering the amount of NDGA detected in the aqueous extract of the plant, at the concentrations analyzed in this case, antiproliferative activity of Larrea divaricata cannot be attributed to this compound, but could have an additive effect on the activity of other compounds.     

Metabolic dependence of ionic channel function studied in the nerve cell membrane

The action of a change in the intracellular 3,5-cAMP (cAMP) level on steady-state and potential-dependent transmembrane ionic currents was investigated in vertebrate and invertebrate nerve cells. The change was produced by injecting cAMP directly into the cell or indirectly, by stimulating or inhibiting activity of various enzymes of the cyclase system. An increase in the intracellular cAMP concentration was found to cause activation of the steady-state two-component transmembrane current, the early component of which is linked mainly with an increase in sodium and calcium, the latter with an increase in potassium conductance of the membrane (possibly due to the entry of calcium ions inside the cell). A decrease in the intracellular cAMP concentration (by intracellular dialysis) evokes weakening of the potential-activated inward calcium current, whereas an increase leads to its restoration. Restoration of the calcium current can also be achieved by activation of the intracellular adenylate cyclase, inhibition of phosphodiesterase, or through direct injection of the catalytic subunit of cAMP-dependent protein kinase inside the cell. Evidence is presented that the regulatory effects obtained are mediated through cAMP-dependent phosphorylation of proteins in the corresponding ionic channels. Elevation of the intracellular calcium ion level interacts closely with the regulatory system described above through activation of some of its enzymic processes.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 286–296, May–June, 1984.     

Relationship between calcium,cyclic AMP,ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility

Capacitation of hamster caudal spermatozoa at a density of 1 × 10⁶/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 × 10⁶/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.