Serotonin induced protein phosphorylation in the Aplysia eye

Serotonin (5-HT) increases the phosphorylation of two low molecular weight phosphoproteins of 23,000 and 15,000 daltons molecular weight and decreases the phosphorylation of a 20,000 dalton phosphoprotein in the isolated Aplysia eye. The cAMP analog 8-benzylthio cAMP increases and decreases the phosphorylation of the 23,000 and 20,000 dalton 5-HT sensitive phosphoproteins, respectively. The effect of 5-HT on protein phosphorylation is not affected by the phase of the circadian rhythm of spontaneous compound action potentials generated in the eye.     

Increasing external K+ blocks phase shifts in a circadian rhythm produced by serotonin or 8-benzylthio-cAMP

Serotonin (5-HT) phase shifts the circadian rhythm from the isolated eye of Aplysia. The discovery of the mechanisms involved in phase shifting by 5-HT may help elucidate the nature of the circadian oscillator. We have found that 5-HT appears to phase shift by causing a change in membrane K+ conductance. Solutions containing zero K+(0-K+) phase shift the rhythm and the phase response curve (PRC) for 0-K+ is similar to one previously obtained for 5-HT. The similarity in PRCs for 0-K+ and 5-HT suggested that these treatments may be phase shifting the rhythm through a common mechanism. The nonadditivity of phase shifting by 0-K+ and 5-HT supports this suggestion. A common mechanism of action of 5-HT and 0-K+ might be effects on membrane potentials. The possible involvement of a membrane potential change in mediating the effect of 5-HT and the lack of an effect of large reductions in Na+, Cl-, and Ca2+ ions on phase shifting by 5-HT led us to examine the role of K+ ions in phase shifting by 5-HT. A change in K+ conductance may mediate the effects of 5-HT on the rhythm because HiK (30mM) solutions blocked the phase shift normally produced by 5-HT. The conductance change produced by 5-HT may be an increase in K+ conductance which would produce a hyperpolarization and not a decrease in K+ conductance which would produce a depolarization since depolarizing treatments, HiK (30-110mM), had no effect on the rhythm at the phase where 5-HT produces its largest phase shifts. Since we previously found that the effects of 5-HT appear to be mediated by cAMP, we examined whether HiK solutions could block the effects of 8-benzylthio-cAMP on the rhythm. HiK (40mM) blocked the phase shifts normally produced by 8-benylthio-cAMP. Our working hypothesis for the 5-HT phase-shifting pathway based on these results is 5-HT leads to increased cAMP leads to elevates K+ conductance leads to membrane hyperpolarization leads to phase shifts the rhythm.     

Calcium plays a central role in phase shifting the ocular circadian pacemaker of Aplysia

The eye of the marine mollusk Aplysia californica contains an oscillator that drives a circadian rhythm of spontaneous compound action potentials in the optic nerve. Both light and serotonin are known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate the role of extracellular calcium in both light and serotonin-mediated phase shifts. Low calcium treatments were found to cause phase shifts which resembled those produced by the transmitter serotonin. However, unlike serotonin, low calcium neither increased ocular cAMP levels nor could these phase shifts be prevented by increasing extracellular potassium concentration. Low calcium-induced phase shifts were prevented by the simultaneous application of the translational inhibitor anisomycin and low calcium treatment resulted in changes in [³⁵S]methionine incorporation into several proteins as measured by a two-dimensional electrophoresis gel analysis. Finally, light treatments failed to produce phase shifts in the presence of low calcium or the calcium channel antagonist nickel chloride. These results are consistent with a model in which serotonin phase shifts the ocular pacemaker by decreasing a transmembrane calcium flux through membrane hyperpolarization while light-induced phase shifts are mediated by an increase in calcium flux.Abbreviations ASW artificial seawater - EGTA ethylene glycol-bis(-amino-ethyl ester) N,N,N N-tetraacetic acid - CAP compound action potential - CT circadian time 5-HT serotonin - Ni⁺⁺ nickel     

Twenty-four hour oscillation of cAMP in chick pineal cells: role of cAMP in the acute and circadian regulation of melatonin production

Chick pineal cells contain circadian oscillators that regulate a rhythm of melatonin biosynthesis. We explored the role of cAMP in regulating this melatonin rhythm. Chick pineal cells expressed a 24 hr oscillation of cAMP efflux with a waveform similar to that of melatonin. Elevation of cAMP in chick pineal cells stimulated melatonin. These results suggest that an oscillation of cAMP regulates the rhythm of melatonin. We investigated whether cAMP was a component of the circadian oscillator by determining the effects of 8-Br cAMP pulses on the phase of the circadian melatonin rhythm. Six hour pulses of 8-Br cAMP did not cause steady-state phase shifts of the rhythm. The acute regulation of melatonin by cAMP, the 24 hr oscillation of cAMP, and the inability of cAMP to phase-shift the melatonin rhythm strongly suggest that cAMP acts as an output signal of the circadian oscillator.     

Evidence that potassium channels mediate the effects of serotonin on the ocular circadian pacemaker of Aplysia

Summary The eye of the marine mollusk Aplysia californica contains a photo-entrainable circadian pacemaker that drives an overt circadian rhythm of spontaneous compound action potentials in the optic nerve. Serotonin is known to influence the phase of this ocular rhythm. The aim of the present study was to evaluate whether potassium channels are involved in effects on the ocular circadian rhythm. Our experimental approach was to study the effect of the potassium channel antagonist barium on serotonin-induced phase shifts of this rhythm. The application of barium was found to block serotonininduced phase shifts whereas barium alone did not cause significant phase shifts. The effects of barium were found to be dose dependent. In addition, barium blocked forskolin-induced phase advances but did not interfere with serotonin-induced increases in cAMP content. Finally, barium antagonized serotonin-induced suppression of compound action potential activity. These results are consistent with a model in which the application of serotonin phase shifts the ocular pacemaker by causing a membrane hyperpolarization which is mediated by a cAMP-dependent potassium conductance.Abbreviations ASW artificial seawater - Ba^{+ +} barium - CAP compound action potential - CT circadian time - 5-HT serotonin - TEA tetraethylammonium     

Cyclic nucleotide- and calcium-independent phosphorylation of proteins in rat brain polyribosome: Effects of ACTH,spermine, and hemin

The incorporation of [-³²P]ATP into proteins of rat brain polyribosomes was studied in vitro. The effects of cyclic nucleotides, calcium, hemin, ACTH, GTP, and spermine were examined. The incorporation of phosphate into proteins increased with time and phosphatase activity was very low; thus, the extent of phosphorylation was predominantly a reflection of protein kinase activity. Phosphorylation of proteins was not sensitive to Ca²⁺ in the presence or absence of either calmodulin or phosphatidylserine. Phosphorylation was also unaffected by cyclic nucleotides in the absence of exogenous enzymes. However, addition of a cMAP-dependent protein kinase together with cAMP resulted in a stimulation of the incorporation of phosphate into 4 phosphoproteins (pp70, pp58, pp43, and pp32); phosphorylation of pp32 was completely dependent on the addition of the kinase. ACTH (1–24), (11–24), and spermine inhibited the endogenous phosphorylation of one protein band (pp30). The phosphorylation of this 30 kD band was also selectively increased by hemin (5 M). Higher concentrations of hemin exerted an inhibitory effect on the majority of the phosphoproteins. Protein phosphatase activity was not influenced by ACTH or spermine. The specific inhibition of pp30 phosphorylation by ACTH or spermine is most probably explained by an interaction with a cyclic nucleotide- and Ca²⁺-independent protein kinase.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

Possible involvement of nitric oxide in generation of orcadian rhythm

Abstract

The effect of continuous infusion of N^G‐methylarginine, an inhibitor of nitric oxide synthase, into the third cerebral ventricle above the suprachiasmatic nucleus (SCN) of the hypothalamus on the circadian rhythm of water intake was examined in rats maintained under either a 12‐h light and 12‐h dark cycle or in constant darkness. N^G‐methylarginine disrupted the circadian rhythm in both conditions. In constant darkness, however, the effect of the inhibitor on the rhythm was found to be due to change in its phase. These findings suggest that nitric oxide is involved in the mechanism of the generation and/or synchronization of the circadian rhythm driven by the circadian oscillator in the SCN.     

Effects of adenylates on the circadian interaction of KaiB with the KaiC complex in the reconstituted cyanobacterial Kai protein oscillator

Cyanobacteria are photosynthetic prokaryotes that possess circadian oscillators. Clock proteins, KaiA, KaiB, KaiC compose the central circadian oscillator, which can be reconstituted in vitro in the presence of ATP. KaiC has ATPase, autokinase, and autophosphatase enzymatic activities. These activities are modulated by protein–protein interactions among the Kai proteins. The interaction of KaiB with the KaiC complex shows a circadian rhythm in the reconstituted system. We previously developed a quantitative, real-time monitoring system for the dynamic behavior of the complex using fluorescence correlation spectroscopy. Here, we examined the effects of ATP and ADP on the rhythmic interaction of KaiB. We show that increased concentration of ATP or ADP shortened period length. Adding ADP to the Kai protein oscillation shifted its phase in a phase-dependent manner. These results provide insight into how circadian oscillation entrainment mechanism is linked to cellular metabolism.     

A comparative study of the effect of abscisic acid and cAMP on protein synthesis in wheat caryopses under drought conditions

The effect of exogenous abscisic acid and cAMP on synthesis of soluble proteins in wheat caryopses in drought has been studied. Both compounds affected the formation of the polypeptides whose synthesis was stimulated by dehydration: they increased the incorporation of the label into polypeptides of 13, 15, and 26 kD and decreased the incorporation of the label into polypeptides of 14, 64, and 77 kD. Abscisic acid and cAMP increased the level of the incorporation of [¹⁴C]leucine into the low-molecular-weight polypeptides of 12, 17, and 19 kD whose synthesis was suppressed by drought. These data suggest that the cyclic adenylate signal system is probably involved in the effect of abscisic acid on protein synthesis in drought.     

Circadian clock regulation of the bimodal rhythm of cyclic AMP in wild-type Euglena

Cell-cycle traverse is associated with fluctuations in the cellular content of cAMP; artificial alterations of these levels phase-shift cell division in free-running cultures of achlorophyllous Euglena maintained in constant darkness (DD). The phase shifts observed, however, are only transient: the cell division rhythm rephases to that of unperturbed controls. This implies that the second messenger functions downstream of the circadian oscillator. Further, the level of cAMP is known to indicate carbon nutrient status and the competency of cells to traverse various restriction points in the cell cycle of other eukaryotes. We wished to determine the profile of cAMP content in free-running, dividing and non-dividing cultures of green, wild-type cells, which survive well during prolonged growth arrest. We monitored cAMP content in photoautothropic cultures of E. gracilis (strain Z) at 25 degrees C under either an entraining light-dark cycle comprising 12 h of light and 12 h of darkness (LD:12,12) or free-running (LD:1/2,1/2) regimes. cAMP content in rhythmically dividing, light-phased or free-running cells exhibited bimodality [peaks at CT (circadian time) 9-14 and CT 19-22). Expression of cAMP content on a per milligram total cellular protein basis caused the day trough (CT 1-3) to be even more distinct. Non-dividing, free-running, photoautotrophic cultures displayed a similarly phased bimodality in cAMP content. These findings in wild-type Euglena confirm that the bimodal rhythm of cAMP content is regulated by the circadian oscillator that underlies division rhythmicity but is not dependent on the cell division cycle. We will now determine the effect of the fluctuating cAMP levels on the phosphorylation status and activity of cell-cycle regulatory proteins.     

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.     

Comparison of Phase Shifts of the Circadian Rhythm of K Uptake in Lemna gibba G3 by Various Amino Acid Analogs

Phase shifts of the circadian rhythm of K⁺ uptake by Lemna gibba G3, caused by pulse administration of various amino acids analogs, were examined and compared. The various phase shifts were not due to any disturbance in the biosynthesis of amino acids, since the effective time of day and direction of the phase shift caused by analogs were not correlated with the standard amino acid which was modified. Effective analogs could be classified into three groups. The first group was effective during the middle subjective day and caused both advances and delays in phase. The second group was effective early in the subjective night, causing large delays and small phase advance. Analogs in the third group shifted the phase as did cycloheximide and were effective at the subjective dawn. Since the analogs of the third group were known to inhibit protein synthesis, it is likely that they shift the phase by lowering the level of some protein(s) important for the clock. By contrast, since the analogs in groups 1 and 2 are known to generate abnormal proteins, the different phase-shifting patterns caused by analogs in groups 1 and 2 suggest that at least two other proteins are important for the circadian timing loop. The amino acid analogs shift the phase as a result of their incorporation into these proteins instead of the standard amino acid. This probably alters the structure and/or activities of these proteins.     

Inhibitors of serine/threonine phosphoprotein phosphatases alter circadian properties in Gonyaulax polyedra.

Protein serine/threonine phosphatases were implicated in the regulation of circadian rhythmicity in the marine dinoflagellate Gonyaulax polyedra based on the effects of three inhibitors specific for protein phosphatases 1 and 2A (okadaic acid, calyculin A, and cantharidin). Chronic exposure to okadaic acid resulted in a significant period lengthening, as measured by the bioluminescent glow rhythm, whereas cantharidin and calyculin A caused large phase delays but no persistent effect on period. Short pulses of the phosphatase inhibitors resulted in phase delays that were greatest near subjective dawn. Unlike 6-dimethylaminopurine, a protein kinase inhibitor, okadaic acid, calyculin A, and cantharidin did not block light-induced phase shifts. The inhibitors tested also increased radiolabeled phosphate incorporation into Gonyaulax proteins in vivo and blocked protein phosphatase 1 and 2A activities in Gonyaulax extracts. This study indicates that protein dephosphorylation catalyzed by protein serine/threonine phosphatases is necessary for proper functioning of the circadian system.     

Neurophysiological mechanisms involved in photo-entrainment of the circadian rhythm from the Aplysia eye

An attempt was made to identify the neurophysiological processes involved in entrainment of the circadian rhythm of spontaneous optic nerve potentials from the Aplysia eye by determining whether pharmacological agents or ion substitutions could block phase shifts produced by single light pulses. Knowing which physiological processes are involved in entrainment should help define the morphological pathway traveled by entrainment information. A secretory step does not appear to be involved in the flow of entrainment information from the environment to the circadian oscillator. A treatment (HiMg LoCa) capable of inhibiting secretion did not interfere with phase shifting by light. Furthermore, treating eyes with putative transmitters or extracts of eyes did not phase shift the free running rhythm. Also, the phase shifting information is not translated into action potentials before reaching the oscillator since TTX–HiMg LoCa solutions did not block the light-induced phase shift. The photoreceptor potential does seem to be important for light-induced phase shifts. A correlation was found between the effects of treatments on the ERG and their effects on the light-induced phase shift. Solutions which decreased the ERG by 90% or more blocked phase shifting whereas solutions which decreased the ERG by less than 74% had no effect on phase shifting by light. The results from these studies are consistent with two pathways for the flow of phase shifting information to the circadian oscillator. The circadian oscillator may be associated with receptor cells and the entrainment pathway would include a step involving the photoreceptor potential. Alternatively, the circadian oscillator may be associated with secondary cells and receive entrainment information via the photoreceptor potential and passive spread of current through a gap junction. Higher order cells than second-order ones are probably not involved in the entrainment pathway.     

In vitro regulation of circadian phosphorylation rhythm of cyanobacterial clock protein KaiC by KaiA and KaiB

Biochemical circadian oscillation of KaiC phosphorylation, by mixing three Kai proteins and ATP, has been proven to be the central oscillator of the cyanobacterial circadian clock. In vivo, the intracellular levels of KaiB and KaiC oscillate in a circadian fashion. By scrutinizing KaiC phosphorylation rhythm in a wide range of Kai protein concentrations, KaiA and KaiB were found to be “parameter-tuning” and “state-switching” regulators of KaiC phosphorylation rhythm, respectively. Our results also suggest a possible entrainment mechanism of the cellular circadian clock with the circadian variation of intracellular levels of Kai proteins.     

Identification of CDK- and cyclin-like proteins in the eye of Bulla gouldiana

The ocular circadian rhythm in the eye of Bulla gouldiana is generated by a rhythm in membrane potential of retinal neurons that is driven by alterations in potassium conductance. Since potassium conductance may be modulated by the phosphorylation of potassium channels, the circadian rhythm may reflect rhythmic changes in protein kinase activity. Furthermore, the circadian rhythm recorded from the Bulla eye can be phase shifted by agents that affect protein synthesis and protein phosphorylation on tyrosine residues. Interestingly, the eukaryotic cell division residues. Interestingly, the eukaryotic cell division cycle is generated by similar processes. Rhythmic cell division is regulated by periodic synthesis and degradation of a protein, cyclin, and periodic tyrosine phosphorylation of a cyclin-dependent kinase (cdk), p34^cdc2. The interaction between these two proteins results in rhythmic kinase activity of p34^cdc2. Both cyclin and p34^cdc2 are pat of two diverse gene families, some of whose members have been localized to postmitotic cell types with no function yet determined. In the current work, we identify proteins similar to the cdks and cyclin in the eye of Bulla. Neither of these ocular proteins are found in mitotic cells in Bulla, and the cdk-like protein (p40) is specific to the eye. Furthermore, the concentration of the cyclin-like protein (p66) is affected by treatments that phase shift the circadain rhythm. The identification of cdk and cyclin-like proteins in the Bulla eye is consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in Bulla is related to the biochemical mechnism that regulates the eukaryotic cell division cycle. 1994 John Wiley & Sons, Inc.