Densin-180 forms a ternary complex with the (alpha)-subunit of Ca2+/calmodulin-dependent protein kinase II and (alpha)-actinin.

Densin-180 is a transmembrane protein that is tightly associated with the postsynaptic density in CNS neurons and is postulated to function as a synaptic adhesion molecule. Here we report the identification of the alpha-subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and alpha-actinin-4 as potential binding partners for the densin-180 intracellular segment. We demonstrate by yeast two-hybrid and biochemical assays that the intracellular portion of densin-180, the alpha-subunit of CaMKII (CaMKIIalpha), and alpha-actinin interact with each other at distinct binding sites and can form a ternary complex stabilized by multiple interactions. Densin-180 binds specifically to the association domain of CaMKIIalpha and does not bind with high affinity to holoenzymes of CaMKII that contain beta-subunit. The PDZ (PSD-95, DIg, Z0-1) domain of densin contributes to its binding to alpha-actinin. A distinct domain of alpha-actinin interacts with the kinase domains of both alpha- and beta-subunits of CaMKII. Autophosphorylation of CaMKII increases its affinity for densin-180 from an EC(50) of >1 micrometer to an EC(50) of <75-150 nM. In contrast, phosphorylation of densin-180 by CaMKII at serine-1397 only slightly decreases its affinity for CaMKII. The specific interaction of densin-180 with holoenzymes of CaMKII containing only alpha-subunit and the increased affinity of CaMKII for densin-180 after autophosphorylation suggest that densin-180 may be involved in localization of activated CaMKII synthesized in dendrites.

Identification of proteins in the postsynaptic density fraction by mass spectrometry.

Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed the identification of most major proteins in the PSD fraction with the use of an analytical method based on mass spectrometry coupled with searching of the protein sequence databases. At least one protein in each of 26 prominent protein bands from the PSD fraction has now been identified. We found 7 proteins not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog of the yeast septin protein cdc10, which is important for bud formation in yeast. Both myosin-Va and cdc10 are threefold to fivefold enriched in the PSD fraction over brain homogenates. Immunocytochemical localization of myosin-Va in cultured hippocampal neurons shows that it partially colocalizes with PSD-95 at synapses and is also diffusely localized in cell bodies, dendrites, and axons. Cdc10 has a punctate distribution in cell bodies and dendrites, with some of the puncta colocalizing with PSD-95. The results support a role for myosin-Va in transport of materials into spines and for septins in the formation or maintenance of spines.

Activity of cyclic AMP phosphodiesterases and adenylyl cyclase in peripheral nerve after crush and permanent transection injuries.

Recent studies demonstrate that cAMP levels are tightly controlled during demyelination and remyelination in Schwann cells as cAMP decreases to 8-10% of normal following both sciatic nerve crush or permanent transection injury and only begins to increase in the crushed nerve after remyelination (Poduslo, J. F., Walikonis, R. S., Domec, M., Berg, C. T., and Holtz-Heppelmann, C. J. (1995) J. Neurochem. 65, 149-159). To investigate the mechanisms responsible for this change in cAMP levels, cAMP phosphodiesterase (PDE) and adenylyl cyclase activities were determined before and after sciatic nerve injury. Basal cAMP PDE activity in soluble endoneurial homogenates of normal nerve was 34.9 +/- 1.9 pmol/mg of protein/min (chi +/- S.E.; n = 10). This activity increased about 3-fold within 6 days following both injuries. Basal PDE activity remained elevated in the transected nerve, but declined to 70 pmol/mg of protein/min in the crushed nerve at 21 and 35 days following injury. Isozyme-specific inhibitors and stimulators were used to identify the PDE families in the sciatic nerve. The low Km cAMP-specific (PDE4) and the Ca2+/calmodulin-stimulated (PDE1) families were found to predominate in assays using endoneurial homogenates. The PDE4 inhibitor rolipram also increased cAMP levels significantly after incubation of endoneurial tissue with various isozyme-specific inhibitors, indicating that PDE4 plays a major role in determining cAMP levels. PDE4 mRNA was localized by in situ hybridization to cells identified as Schwann cells by colabeling of S100, a Schwann cell specific protein. Adenylyl cyclase activity declined following injury, from 3.7 pmol/mg of protein/min in normal nerve to 0.70 pmol/mg/min by 7 days following injury. Both decreased synthesis and increased degradation contribute, therefore, to the reduced levels of cAMP following peripheral nerve injury and are likely critical to the process of Wallerian degeneration.

The second messenger, cyclic AMP, is not sufficient for myelin gene induction in the peripheral nervous system.

The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8-10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P0 mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P0 gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.

Excretion of catecholamines and metabolites in response to increased dietary phosphate intake.

The urinary excretion of free dopamine, norepinephrine, and epinephrine could reflect the contribution of the neural release and filtration of these catecholamines as well as the intrarenal tubular synthesis and metabolism of dopamine. Because these catecholamines are rapidly metabolized, the excretion of the free amines represents only a fraction of the total release and synthesis by the kidney. The present study determined the effect of increasing dietary phosphate intake on the excretion of free dopamine, norepinephrine, and epinephrine and their primary stable metabolites. Seven male rats were placed in metabolic balance cages and fed 12 gm/day of normal phosphate diet (NPD) (0.7% inorganic phosphorus [Pi]) for 4 days and then fed a high phosphate diet (HPD) (1.8% Pi) for 4 days. Twenty-four-hour urine samples were collected for determination of free catecholamines, their major stable metabolites, and electrolyte excretions. The urinary excretion data for the seven rats was combined for all 4 days of each dietary regimen. Increasing phosphate intake from 0.7% to 1.8% significantly increased free dopamine excretion by 23%, from 5.6 +/- 0.2 to 6.8 +/- 0.1 micrograms/day (n = 7, p < 0.05). This increase in free dopamine excretion was associated with similar increases in urinary excretion of dopamine glucuronide, 21.6 +/- 1.3 to 27.9 +/- 1.8 micrograms/day (32%) and the dopamine metabolite DOPAC, 9.4 +/- 0.5 to 12.1 +/- 0.6 micrograms/day (30%) and total dopamine excretion from 32.9 +/- 1.7 to 41.0 +/- 1.9 micrograms/day (27%). Plasma DOPA levels were unchanged by increased dietary phosphate intake; however, plasma norepinephrine levels decreased significantly. Excretion of free or sulfated norepinephrine was not changed by increased phosphate intake. However, excretion of MHPG, a metabolite of norepinephrine and epinephrine, decreased significantly, from 33.7 +/- 2.1 to 23.9 +/- 0.8 micrograms/day, n = 7, p < 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Attractiveness of the male Acheta domesticus calling song to females. III. The relation of age-correlated changes in syllable period recognition and phonotactic threshold to juvenile hormone III biosynthesis.

1. Most crickets first demonstrated positive phonotaxis to 65 dB CSs having a 53-62 ms SP by day 3 following the imaginal molt (Fig. 3B). The onset of copulatory readiness occurred on average at 3.2 days. 2. The attractive range of SPs for most females became progressively broader as they aged (Fig. 4). Three to 4-day-old females were attracted to a smaller number of CS SPs than were 20-21 day old females (Fig. 4). 3. Older, less selective females did not typically respond to the same range of CS SPs (Fig. 6). However, they were more likely to respond to some SPs (especially 50 ms) than to others (Fig. 7). 4. The phonotactic threshold decreased from 95 dB or greater on day 0 to a mean of 55 dB by day 3, during a period of increasing JHIII biosynthesis, and thereafter remained at that level (Fig. 8). 5. During a period of maximal JHIII production, 3-5 day-old females usually responded to 4 of the 7 SPs presented (Fig. 8). Females older than 12 days were unselective for CS SP, and JHIII synthesis remained at a level below the peak production on day 4 (Fig. 8). 6. Older females, that were unselective for CS SP, became as selective as 3 to 5-day-old females within 4 days of topical application of JHIII (Figs. 9-11).