Cannabinoid receptor agonists inhibit depolarization-induced calcium influx in cerebellar granule neurons.

Neuronal cannabinoid receptors (CB(1)) are coupled to inhibition of voltage-sensitive Ca(2+) channels (VSCCs) in several cell types. The purpose of these studies was to characterize the interaction between endogenous CB(1) receptors and VSCCs in cerebellar granule neurons (CGN). Ca(2+) transients were evoked by KCl-induced depolarization and imaged using fura-2. The CB(1) receptor agonists CP55940, Win 55212-2 and N-arachidonylethanolamine (anandamide) produced concentration-related decreases in peak amplitude of the Ca(2+) response and total Ca(2+) influx. Pre-treatment of CGN with pertussis toxin abolished agonist-mediated inhibition. The inhibitory effect of Win 55212-2 on Ca(2+) influx was additive with inhibition produced by omega-agatoxin IVA and nifedipine but not with omega-conotoxin GVIA, indicating that N-type VSCCs are the primary effector. Paradoxically, the CB(1) receptor antagonist, SR141716, also inhibited KCl-induced Ca(2+) influx into CGN in a concentration-related manner. SR141716 inhibition was pertussis toxin-insensitive and was not additive with the inhibition produced by Win 55212-2. Confocal imaging of CGN in primary culture demonstrate a high density of CB(1) receptor expression on CGN plasma membranes, including the neuritic processes. These data demonstrate that the CB(1) receptor is highly expressed by CGN and agonists serve as potent and efficacious inhibitory modulators of Ca(2+) influx through N-type VSCC.

Drosophila dec-1 eggshell proteins are differentially distributed via a multistep extracellular processing and localization pathway.

In Drosophila the multilayered eggshell forms during late oogenesis between the oocyte and the overlaying follicle cells. Proper eggshell assembly requires wild-type dec-1 gene function. Alternatively spliced dec-1 transcripts encode three proproteins that are cleaved extracellularly in a stage-specific manner to at least five distinct derivatives. Using polyclonal antibodies raised against fusion proteins containing different regions of the dec-1 proteins, we have localized several dec-1 derivatives in the assembling and completed eggshell. Although all of the dec-1 derivatives are generated in the oocyte proximal vitelline membrane layer, they are differentially distributed in the mature egg. Some derivatives are gradually released from the vitelline membrane and become localized within distinct regions of the chorion, while others are taken up by the oocyte or become concentrated in the endochorionic spaces or cavities. The diverse distributions of the dec-1 derivatives suggest that each derivative plays a distinct role in eggshell assembly. These results also suggest that the vitelline membrane layer, by acting as a transient storage site, may control the availability of molecules active in eggshell assembly and by extension perhaps other follicle cell products important in early embryonic pattern formation.

Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry.

The distribution in the rat brain of fatty acid amide hydrolase (FAAH) an enzyme that catalyzes the hydrolysis of the endogenous cannabinoid anandamide was studied by immunohistochemistry. An immunopurified, polyclonal antibody to the C terminal region of FAAH was used in these studies. The large principal neurons, such as pyramidal cells in the cerebral cortex, the pyramidal cells the hippocampus, Purkinje cells in the cerebellar cortex and the mitral cells in the olfactory bulb, showed the strongest FAAH immunoreactivity. These FAAH-containing principal neurons except the mitral cells in the olfactory bulb are in close proximity with cannabinoid CB1 receptors as revealed by our previous immunohistochemical study. Moderately or lightly stained FAAH-containing neurons were also found in the amygdala, the basal ganglia, the deep cerebellar nuclei, the ventral posterior nuclei of the thalamus, the optic layer and the intermediate white layer of the superior colliculus and the red nucleus in the midbrain, and motor neurons of the spinal cord. These data demonstrate that FAAH is heterogeneously distributed and this distribution exhibits considerable, although not complete, overlap with the distribution of cannabinoid CB1 receptors in rat brain.

Regulated processing of dec-1 eggshell proteins in Drosophila.

The Drosophila dec-1 gene encodes multiple products that are necessary for proper eggshell assembly. During stages 9-12 of oogenesis the ovarian follicle cells synthesize three alternatively spliced dec-1 RNAs that encode proteins of 106, 125, and 177 kDa. All three of these primary translation products undergo developmentally regulated posttranslational cleavages. Antibodies to trpE fusion proteins containing different regions of the dec-1 proteins have been used to identify processing intermediates as well as stable derivatives of fc106, fc125, and fc177. fc106, the most abundant product of the locus, is cleaved at its N-terminus, producing an N-terminal 25-kDa derivative and s80, a stage 10 eggshell protein. During late oogenesis N-terminal cleavage of s80 yields a 20-kDa N-terminal derivative and a 60-kDa eggshell protein. fc125, which differs from fc106 by a C-terminal extension, is processed at its N-terminus through a series of transient intermediates to a stable 95-kDa C-terminal derivative in late stage 10 egg chambers. Unlike its s80 counterpart, the 95-kDa derivative is not subjected to later cleavage events. fc177, synthesized during stages 11-12, is cleaved to a stable C-terminal derivative of approximately 85 kDa. Although all of the cleavage sites fall within regions that are common to fc106, fc125, and fc177, each protein follows a different maturation pathway which results in a variety of proteins with distinctive N- and C-termini. Our data suggest that inter- or intramolecular interactions dictated by the C-terminal ends of the molecules determine the pathway followed by each dec-1 protein.