Modulation of cardiac beta-adrenergic receptors by dopamine beta-hydroxylase

Incubation of cardiac sarcolemma in the presence of dopamine beta-hydroxylase (DBH), a catecholamine biosynthetic enzyme, increased beta-adrenergic receptor density by 68% as measured by [3H]dihydroalprenolol (DHA) binding. The addition of DBH to plasma membranes isolated from brain, kidney, skeletal muscle, liver and intestine did not alter [3H]DHA binding. Cardiac alpha-receptors were unaffected under similar conditions. Since DBH is coreleased with norepinephrine, these results indicate that a functional coupling of the putative beta-adrenergic receptor with DBH may exist in cardiac muscle.     

Modulation of dopamine receptors by thyrotropin-releasing hormone in the rat brain

Nanomolar concentration of thyrotropin-releasing hormone (TRH) in vitro caused a significant reduction of [3H]apomorphine binding sites (70% of the control) in the rat striatum and the limbic forebrain. [3H]Spiperone binding was not affected by TRH. On the other hand, dopamine and apomorphine displaced [3H]TRH binding partially, suggesting the presence of a TRH receptor subpopulation that has a high affinity for dopamine agonist. Most of the neuroleptics displaced [3H]TRH binding dose-dependently in the micromolar range. (-)-Sulpiride had no affinity to TRH receptors. These findings suggest that one of the important roles of TRH as a neuromodulator is to modulate receptors for classical neurotransmitters, and this receptor-receptor interaction may be of importance in explaining the well known stimulating effects of TRH on the dopaminergic system.     

Molecular evolution and recombination in gender-associated mitochondrial DNAs of the Manila clam Tapes philippinarum

Doubly uniparental inheritance (DUI) provides an intriguing system for addressing aspects of molecular evolution and intermolecular recombination of mitochondrial DNA. For this reason, a large sequence analysis has been performed on Tapes philippinarum (Bivalvia, Veneridae), which has mitochondrial DNA heteroplasmy that is consistent with a DUI. The sequences of a 9.2-kb region (containing 29 genes) from 9 individuals and the sequences of a single gene from another 44 individuals are analyzed. Comparisons suggest that the two sex-related mitochondrial genomes do not experience a neutral pattern of divergence and that selection may act with varying strength on different genes. This pattern of evolution may be related to the long, separate history of M and F genomes within their tissue-specific "arenas." Moreover, our data suggest that recombinants, although occurring in soma, may seldom be transmitted to progeny in T. philippinarum.     

Cellular defence mechanism of the clam Tapes semidecussatus against infection by the protozoan Perkinsus sp.

The cellular defence mechanism of the clam Tapes semidecussatus (Mollusca, Bivalvia) against infection by the parasite protozoan Perkinsus sp. (Apicomplexa, Perkinsea) was studied in the gill filaments. The parasites, localized in the connective tissue, induced a cellular reaction involving infiltrated granulocytes. These showed a secretory aspect, with the cytoplasm being filled by membrane-bound granules with internal membranes. The holocrine secretion, which was proteic and slightly glycosylated, by the granulocytes gave rise to the encapsulation of the parasites. After incubation with lectins from Canavalia ensiformis, Triticum vulgaris, Helix pomatia, Glycine max, Arachis hypogaea, Ricinus communis (agglutinin), Ulex europeus I and Limax flavus, a lack of specific and/or main sugars was observed in the plasma membrane of parasite and granulocyte, and in the wall of the former. Furthermore, GalNac1,3GalNac and -d-gal residues were only detected in association with the internal membranes and dense regions of both granules and capsule, respectively. Blood granulocytes were observed at the periphery of the cellular reaction, close to blood vessels, and these appeared to re-differentiate to give the granulocytes of the cellular reaction. The data reported here suggest that this parasite induces the infiltration and re-differentiation of specialized cells in the host mollusc. In addition, a polarized secretion of a specific defence product is described for the first time.     

First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum

In the present study, we report on haemocyte distribution, determined by a Coulter Counter, in the clam Tapes philippinarum. In addition, cytoskeleton components of haemocytes were examined using specific probes for F-actin and alpha-tubulin. The mean number of circulating haemocytes was 5 (x10(6))cells/ml haemolymph. Two main haemocyte populations were found in the haemolymph: small cells, 2-3microm in diameter and 10-100fl in volume; and large cells, 6-10microm in diameter and 150-400fl in volume. Analysis of the haemocyte cytoskeleton revealed bundles of actin filaments oriented according to the cell major axis, and microtubules radiating from the microtubule-organizing centre in proximity of the nucleus. Interestingly, mitotic spindles were also found radiating from the microtubule-organizing centres, located at the spindle poles (centrosomes) of undifferentiated cells. On the basis of both our previous findings regarding circulating stem cells (Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol 10, 677-693) and new information from the present study, we suggest that haemoblasts are able to divide in the haemolymph of T. philippinarum. To our knowledge, this is the first report of mitotic spindles in circulating haemocytes from a bivalve species.     

Modulation of adenyl cyclase activity in the gills of Tapes philippinarum

Adenyl cyclase (AC) plays a pivotal role in cell signaling. The AC system of bivalves has received little attention so far, and our study has been addressed to the characterization of AC properties in the gills of T. philippinarum. The enzyme showed a Km value of 0.77 mM for ATP in the presence of 5 mM Mg2+; in the absence of agonists, it was poorly affected by GTP, while it was stimulated by GTPgammaS and GppNHp up to 14-fold and 4-fold, respectively. Similarly to other invertebrates, the enzyme activity was scarcely stimulated by forskolin. The receptor agonist serotonin (5-HT) significantly stimulated the AC activity, and the pharmacological profile of the 5-HT receptor/s was as follows: (+)butaclamol > dihydroergocryptine > methysergide > prazosin > yohimbine. The AC activity was assessed in vitro in the presence of tributyltin chloride and HgCl2, which reduced the AC activity only at the highest dose tested (10-100 microM). Our data indicate the presence of a membrane-bound AC in gill membranes of T. philippinarum, coupled to Gs proteins and to a specific class of 5-HT receptors. Such receptors show a pharmacological profile slightly different from that reported for 5-HT invertebrate receptors cloned so far.     

Modulation of the SNARE core complex by dopamine

Communication between nerve cells in the brain occurs primarily through specialized junctions called synapses. Recently, many details of synaptic transmission have emerged. The identities of specific proteins important for synaptic vesicle release have now been established. We have investigated three synaptic proteins, VAMP (vesicle associated membrane protein; also called synaptobrevin), syntaxin, and SNAP25 (synaptosomal associated protein of 25kDa) as possible targets in the dopamine-mediated modulation of synaptic function in rat striatal slices. These three proteins form a SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptors) core complex that is known to be essential for synaptic transmission. Although it is envisioned that the SNAREs undergo dynamic and cyclic interactions to elicit synaptic vesicle release, their precise functions in neurotransmission remains unknown. We have examined SNARE complexes in intact rat striatal slices. Cellular proteins were solubilized, separated electrophoretically by SDS-PAGE, and then identified immunologically. Application of dopamine to striatal slices results in SNAREs favoring the SNARE core complex, a complex which forms spontaneously in the absence of crosslinking agents, rather than the monomer form. In addition, rapid crosslinking of dopamine-treated striatal slices demonstrates that the SNARE complex is increased 4 fold in dopamine treated striatal slices compared with control slices. Haloperidol blocked the dopamine-induced change in the core complex. These results suggest that changes in the activities of SNAREs may be involved in the underlying cellular mechanisms(s) of dopamine-regulated synaptic plasticity of the striatum.     

Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticus were established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (< 1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin.     

Modulation of the Aplysia gill withdrawal reflex by dopamine

The ability of dopamine to modulate gill contractions was tested in Aplysia. When dopamine was perfused through the gill vasculature, gill contractions caused by siphon stimulation (gill withdrawal reflex) and by depolarization of the gill motor neuron L7 were increased in amplitude, as compared with those evoked during seawater perfusion. Habituation of gill movements, brought about by repetitive stimulation of the siphon or of L7, was prevented by dopamine. Despite the absence of reflex habituation, the number of action potentials in central gill motor neurons, evoked by siphon stimulation, showed normal decrement. Dopamine's effects were blocked when the ctenidial nerve was cut or when L7 hyperpolarized. These data suggest that dopamine acts peripherally to increase the efficacy of L7's synaptic transmission onto gill muscle or elements of the gill neural plexus.     

Behavioral sensitization and tolerance to cocaine and the occupation of dopamine receptors by dopamine

Data from the authors’ laboratory on the neural substrates of Pavlovian conditioning and behavioral sensitization to psychomotor stimulants are reviewed. The findings of a recent experiment on the role of occupation of dopamine receptors by dopamine and its association to behavioral sensitization are reported. Daily intermittent injections of cocaine produced behavioral sensitization to the locomotor response in rats, whereas continuous cocaine infusions produced behavioral tolerance. Behavioral sensitization to cocaine was blocked by coadministration of nimodipine, anL-type calcium channel blocker. The increases in locomotion produced by cocaine was associated with an increase in the occupation of striatal dopamine D₁ and D₂ receptors, measured as the density of receptors protected from denaturation byN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). This association was not observed when rats were given a challenge injection of cocaine 10 d after withdrawal from similar treatment regimens. Rats given a cocaine challenge after withdrawal from either intermittent or continuous cocaine treatment regimens exhibited increased occupations of striatal D₁ and D₂ receptors. This increase was similar in magnitude to that observed in rats without a history of cocaine treatments after a challenge injection of cocaine. This suggests tnat the differences in occupancy of striatal dopamine receptors by dopamine observed in the prewithdrawal condition are likely the result of differences in brain levels of cocaine achieved by the two treatment regimens. Occupancy of striatals dopamine D₁ and D₂ receptors does not appear to be related to the development of sensitization to the motor-stimulating effects of cocaine.     

Modulation of the cannabinoid receptors by hemopressin peptides

Changes in the endocannabinoid system are implicated in numerous diseases, making it an attractive target for pharmaceutical development. The endocannabinoid receptors have traditionally been thought to act through the effects of lipophilic messengers called cannabinoids. The exciting finding of endocannabinoid system modulation by the nonapeptide hemopressin and its N-terminal extensions has highlighted the complexity of cannabinoid biology and pharmacology and sparked interest for therapeutic purposes. However, many questions surrounding the generation and regulation of the hemopressin peptides, the self-assembly of hemopressin and the potential for drug development based on hemopressin remain and are discussed in this review.     

Modulation of motor behavior by dopamine and the D1-like dopamine receptor AmDOP2 in the honey bee

Determining the specific molecular pathways through which dopamine affects behavior has been complicated by the presence of multiple dopamine receptor subtypes that couple to different second messenger pathways. The observation of freely moving adult bees in an arena was used to investigate the role of dopamine signaling in regulating the behavior of the honey bee. Dopamine or the dopamine receptor antagonist flupenthixol was injected into the hemolymph of worker honey bees. Significant differences between treated and control bees were seen for all behaviors (walking, stopped, upside down, grooming, flying and fanning), and behavioral shifts were dependent on drug dosage and time after injection. To examine the role of dopamine signaling through a specific dopamine receptor in the brain, RNA interference was used to reduce expression levels of a D1-like receptor, AmDOP2. Injection of Amdop2 dsRNA into the mushroom bodies reduced the levels of Amdop2 mRNA and produced significant changes in the amount of time honey bees spent performing specific behaviors with reductions in time spent walking offset by increases in grooming or time spent stopped. Taken together these results establish that dopamine plays an important role in regulating motor behavior of the honey bee.     

Characterization and localization of an Mr 225 kDa polypeptide specifically involved in the defence mechanisms of the clam Tapes semidecussatus

Parasitosis by the trophozoite protozoan Perkinsus sp. (Apicomplexa, Perkinsea) induces in the gill filaments of the clam Tapes semidecussatus (Mollusca, Bivalvia) a cellular reaction, which is constituted by infiltrated granulocytes. This cellular reaction has characteristics of those of a holocrine gland, since the parasites are encapsulated by the secretion product of the granulocytes after cell death. An enriched fraction of prezoosporangia and their associated capsule was obtained after culture of the parasitized gills in fluid thioglycollate medium. Specific polypeptides from this fraction were separated by SDS-PAGE and isolated for rabbit immunizations. The serum obtained against an Mr 225 kDa polypeptide, revealed its exclusive localization in the capsule and in the granules of the infiltrated granulocytes, thus indicating that this polypeptide is synthesized by these cells and secreted, in a polarized way, around the trophozoites resulting in their encapsulation. Selective deglycosylation of the polypeptide, by Endo H and alkaline -elimination, did not show an effect on its molecular weight or antibody recognition. Furthermore, the absence of the 225 kDa band in the Western-blots of non-parasitized gills indicated the specific association of this polypeptide with the parasitosis. Finally, this is the first tissue-specific factor described in molluscs in relation to defence mechanisms.     

Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). II. Temporal pattern of disease dynamics and association with clam mortality

Temporal dynamics of the infection by Perkinsus olseni in a clam (Tapes decussatus) bed was studied over 5 yr (March 1996 to December 2000). Diagnostic techniques were compared to assess their suitability for epizootiological purposes. A technique based on incubation of 2 gill lamellae in Ray's fluid thioglycollate medium (RFTM) was more sensitive, quicker and cheaper than examination of histological sections. Incubation of the whole-clam soft tissues in RFTM allowed detection of very light infections that were not detected with incubation of only 2 gill lamellae. Nevertheless, the correlation between the infection intensity estimated by both RFTM incubations was high. Infection intensity was significantly and positively correlated with clam size/age. No infected clam smaller than 20 mm was found. There was an annual pattern of infection involving lower mean infection intensity and prevalence in winter and higher values for both variables from spring to autumn, with 2 main annual peaks in spring and late summer-early autumn. This temporal pattern was significantly associated with the seawater temperature. The annual spring peak of infection intensity occurred when seawater temperature was around 15 degrees C. Monthly mortality in the clam bed peaked in spring and summer--after peaks of P. olseni infection intensity and concurrently with high seawater temperature. A comparison of percentage mortality between clams from 2 sources (a perkinsosis-affected and a non-affected area) placed in the same clam bed revealed significantly higher mortality in the clams originating from the perkinsosis-affected area.     

Modulation of serotonin receptors by specific phosphatidylcholines

Treatment of mouse cortical brain membranes with dioleoylphosphatidylcholine produced a large (50%) decrease in serotonin binding sites. The time course for this effect paralleled an increase in oleic acid in membrane phosphatidycholine and an increase in membrane fluidity. “Active Lipid” produced a similar decrease in serotonin binding sites, while fluidizing the membranes even more strongly. Distearoylphosphatidylcholine had no effect on serotonin binding sites or membrane fluidity by itself, but was capable of counteracting both the reduction in binding sites and membrane fluidity produced by “Active Lipid”. The data indicate that specific phosphatidylcholines can have profound effects on serotonin receptors, but a clear picture of the relative importance of membrane fluidity per se versus more specific phospholipid effects will require further investigation.     

Modulation of neuritogenesis by astrocyte muscarinic receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.     

Modulation of NMDA receptors by AKT kinase

The aim of the present work was to assess whether Akt modulates NMDA receptor function in cerebellar neurons in culture. Forskolin increases cAMP and activates Akt and NMDA receptors. In neurons treated with forskolin, intracellular calcium increased to 296 +/- 38% and this was completely prevented by inhibition of Akt. This indicates that, in these neurons, cAMP modulates NMDA receptors via Epac and Akt. Brain derived neurotrophic factor (BDNF) increases phosphorylation (and activity) of Akt to 350 +/- 60% of basal and also potentiates the increase of calcium and in cGMP induced by NMDA. BDNF-induced potentiation of NMDA receptor function is completely prevented by inhibition of PI3 kinase or of Akt. This indicates that BDNF modulates NMDA receptor function via PI3 kinase and Akt. Activation of NMDA receptors also leads to phosphorylation and activation of Akt which, in turn, potentiates NMDA receptor activation. The results reported indicate that when Akt activity increases the activation of NMDA receptors by its agonists also increases. Akt may play important roles in the modulation of NMDA receptor responses by other neurotransmitters and modulators and in the adaptation of NMDA receptor function to the physiological environmental conditions.