Electron-microscopic investigation of the innervation of the pars distalis of the hypophysis in adult Rana temporaria L.

Summary In the pars distalis of the hypophysis of adult Rana temporaria, three types of nerve-fiber profiles were found at two distinct sites, in both lateral parts of the bordering regions of the anterior lobe with the intermediate lobe of the hypophysis. The first type of nerve-fiber profile consists of bundles of very fine axonal elements (diameter: <0.7 m). The second type is formed by larger nerve fibers (diameter up to 4 m) containing a few neurosecretory granules of approximately 100 nm. The third type of nervefiber profile resembles the second type but these nerve fibers make synaptoid contacts on at least two different types of glandular cells. The possible functional significance of these nerve fibers in the pars distalis is discussed.No nerve fibers were found (1) in the central part of the bordering region of the pars distalis with the intermediate lobe, (2) at the bordering region with the median eminence and (3) with the neurohypophysial stalk, and (4) in all other parts of the pars distalis.     

Vascularization of the pars distalis of the hypophysis in the toad,Bufo bufo (L.) (Amphibia,Anura)

Summary The vascularization of the pars distalis of the hypophysis of the toad, Bufo bufo (L.), was studied by the traditional method of injecting a mixture of India-ink and gelatine into the circulatory system of the head via the arteria carotis communis. Further, methyl-methacrylate corrosion casts of the brains were made; the hypothalamo-adenohypophysial region of these corrosion casts was studied with the scanning electron microscope. The results showed that the portal vessels which arise from the median eminence do not supply distinct areas in the pars distalis as is supposed by the point-to-point-hypothesis. The portal vessels enter the ventro-median region of the pars distalis and branch off into a three-dimensional network of the secondary capillary plexus of the pars distalis. The plexus is made up mostly by four- to six-sided meshes. This angioarchitecture guarantees an optimal supply of the glandular cells of the pars distalis with nutritional factors and releasing hormones, on the one hand, and facilitates the removal of the hormones which are released by these cells, on the other hand. The venous drainage of the pars distalis is exerted mainly by two large veins, which bilaterally leave the dorso-lateral region (venous pole) of the pars distalis and by a few small veins, which drain into the wide, sinus-like vessel, which curves around the dorso-caudal region of the pars distalis and joins bilaterally the vena hypophysea transversa.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (Projekt Nr. 2183/N 39) and by the Stiftungs- und Förderungsgesellschaft der Paris-Lodron-Universität SalzburgThe authors wish to thank Mag. Ursula Albrecht for excellent technical assistance and Mr. Gerhard Sulzer for photographic work     

BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus)

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2). The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.     

An electron microscopic study of stellate cells and cavities in the pars distalis of frog pituitary

Stellate cells in the pars distalis of adult Rana ridibunda were observed electron microscopically under normal and experimental conditions (TRH injection). The stellate cells have lengthy processes extending into the intercellular spaces between the secretory cells and scanty cytoplasm surrounding the nucleus. Occasional desmosomes link stellate cells to adjacent secretory cells. In the pars distalis of animals injected with thyrotropic-releasing hormone (TRH), the stellate cells form large cavities (2-6 mum) filled with heterogeneous material. Their cytoplasm contains well-developed Golgi complexes and some lysosomes; these are the principal morphological alterations as compared to those observed in control animals. It is suggested that stellate cells could play an active role in addition to providing a structural framework for the pars distalis.     

Molecular biology of cannabinoid receptors

During the last decade, research on the molecular biology and genetics of cannabinoid receptors has led to a remarkable progress in understanding of the endogenous cannabinoid system, which functions in a plethora of physiological processes in the animal. At present, two types of cannabinoid receptors have been cloned from many vertebrates, and three endogenous ligands (the endocannabinoids arachidonoyl ethanolamide, 2-arachidonoyl glycerol and 2-arachidonoyl-glycerol ether) have been characterized. Cannabinoid receptor type 1 (CB(1)) is expressed predominantly in the central and peripheral nervous system, while cannabinoid receptor type 2 (CB(2)) is present almost exclusively in immune cells. Cannabinoid receptors have not yet been cloned from invertebrates, but binding proteins for endocannabinoids, endocannabinoids and metabolic enzyme activity have been described in a variety of invertebrates except for molting invertebrates such as Caenorhabditis elegans and Drosophila. In the central nervous system of mammals, there is strong evidence emerging that the CB(1) and its ligands comprise a neuromodulatory system functionally interacting with other neurotransmitter systems. Furthermore, the presynaptic localization of CB(1) together with the results obtained from electrophysiological experiments strengthen the notion that in cerebellum and hippocampus and possibly in other regions of the central nervous system, endocannabinoids may act as retrograde messengers to suppress neurotransmitter release at the presynaptic site. Many recent studies using genetically modified mouse lines which lack CB(1) and/or CB(2) finally could show the importance of cannabinoid receptors in animal physiology and will contribute to unravel the full complexity of the cannabinoid system.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Immunofluorescent identification of somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey,Macacus irus

Summary Using antibodies against human growth hormone and ovine prolactine, it is possible to identify somatotropic and prolactin cells in the anterior lobe of the hypophysis (pars distalis) of the monkey, Macacus irus. These cells are present in both adult and infant male and female monkeys. The density of the somatotropic cells is greater at the periphery of the anterior lobe. Prolactin cells are of two types: one type is dispersed throughout the pars distalis and consists of small and large cells, the other type forms clusters of large cells.We wish to express our thanks to Mrs. M. Raccurt (CNRS) for her highly skilled technical assistance     

Cannabinoid receptor ligands mediate growth inhibition and cell death in mantle cell lymphoma

We have earlier reported overexpression of the central and peripheral cannabinoid receptors CB1 and CB2 in mantle cell lymphoma (MCL), a B cell non-Hodgkin lymphoma. In this study, treatment with cannabinoid receptor ligands caused a decrease in viability of MCL cells, while control cells lacking CB1 were not affected. Interestingly, equipotent doses of the CB1 antagonist SR141716A and the CB1/CB2 agonist anandamide inflicted additive negative effects on viability. Moreover, treatment with the CB1/CB2 agonist Win-55,212-2 caused a decrease in long-term growth of MCL cells in culture. Induction of apoptosis, as measured by FACS/Annexin V-FITC, contributed to the growth suppressive effect of Win-55,212-2. Our data suggest that cannabinoid receptors may be considered as potential therapeutic targets in MCL.     

Evidence for the presence of CB1 cannabinoid receptors on peripheral neurones and for the existence of neuronal non-CB1 cannabinoid receptors.

The discovery of CB1 and CB2 receptors and of endogenous agonists for these receptors has sparked renewed interest in the therapeutic potential of cannabinoids. This has led to a need for strategies that will provide a better separation of wanted from unwanted effects, particularly for CB1 receptor agonists. Possible strategies are to target CB1 receptors present on neurones outside the central nervous system or novel types or subtypes of neuronal cannabinoid receptor. This paper reviews evidence for the presence of CB1 receptors on peripheral neurones and for the existence of neuronal non-CB1 cannabinoid receptors.     

Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.     

Delta9-tetrahydrocannabinol (Delta9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia

Delta(9)-tetrahydrocannabinol (Delta(9)-THC), a primary psychoactive constituent of cannabis, has been reported to act as a neuroprotectant via the cannabinoid CB(1) receptor. In this study, Delta(9)-THC significantly decreased the infarct volume in a 4 h mouse middle cerebral artery occlusion mouse model. The neuroprotective effect of Delta(9)-THC was completely abolished by SR141716, cannabinoid CB(1) receptor antagonist, and by warming the animals to 31 degrees C. Delta(9)-THC significantly decreased the rectal temperature, and the hypothermic effect was also inhibited by SR141716 and by warming to 31 degrees C. At 24 h after cerebral ischemia, Delta(9)-THC significantly increased the expression level of CB(1) receptor in both the striatum and cortex, but not in the hypothalamus. Warming to 31 degrees C during 4 h cerebral ischemia did not increase the expression of CB(1) receptor at the striatum and cortex in MCA-occluded mice. These results show that the neuroprotective effect of Delta(9)-THC is mediated by a temperature-dependent mechanism via the CB(1) receptor. In addition, warming to 31 degrees C might attenuate both the neuroprotective and hypothermic effects of Delta(9)-THC through inhibiting the increase in CB(1) receptor in both the striatum and cortex but not in the hypothalamus, which may suggest a new thermoregulation mechanism of Delta(9)-THC.     

Novel Physiologic Functions of Endocannabinoids as Revealed Through the Use of Mutant Mice

The presence in the mammalian brain of specific receptors for marijuana triggered a search for endogenous ligands, several of which have been recently identified. There has been growing in-terest in the possible physiological functions of endocannabinoids, and mutant mice that lack cannabinoid receptors have become an important tool in the search for such functions. To date, studies using CB1 knockout mice have supported the possible role of endocannabinoids in retro-grade synaptic inhibition in the hippocampus, in long-term potentiation and memory, in the de-velopment of opiate dependence, and in the control of appetite and food intake. They also suggested the existence of as yet unidentified cannabinoid receptors in the cardiovascular and central nervous systems. The use of CB2 receptor knockout mice suggested a role for this re-ceptor in macrophage-mediated helper T cell activation. Further studies will undoubtedly reveal many additional roles for this novel signaling system.     

Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities

Human tissues express cannabinoid CB₁ and CB₂ receptors that can be activated by endogenously released ‘endocannabinoids’ or exogenously administered compounds in a manner that reduces the symptoms or opposes the underlying causes of several disorders in need of effective therapy. Three medicines that activate cannabinoid CB₁/CB₂ receptors are now in the clinic: Cesamet (nabilone), Marinol (dronabinol; Δ⁹-tetrahydrocannabinol (Δ⁹-THC)) and Sativex (Δ⁹-THC with cannabidiol). These can be prescribed for the amelioration of chemotherapy-induced nausea and vomiting (Cesamet and Marinol), stimulation of appetite (Marinol) and symptomatic relief of cancer pain and/or management of neuropathic pain and spasticity in adults with multiple sclerosis (Sativex). This review mentions several possible additional therapeutic targets for cannabinoid receptor agonists. These include other kinds of pain, epilepsy, anxiety, depression, Parkinson''s and Huntington''s diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders. It also describes potential strategies for improving the efficacy and/or benefit-to-risk ratio of these agonists in the clinic. These are strategies that involve (i) targeting cannabinoid receptors located outside the blood-brain barrier, (ii) targeting cannabinoid receptors expressed by a particular tissue, (iii) targeting upregulated cannabinoid receptors, (iv) selectively targeting cannabinoid CB₂ receptors, and/or (v) adjunctive ‘multi-targeting’.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Immunocytochemical analysis of somatotrophs and mammotrophs in the pars distalis of postnatal dwarf (dw) mutant mice

Summary The postnatal differentiation of somatotrophs and mammotrophs in the pars distalis of normal and dwarf (dw/dw) mice was analyzed by means of immunocytochemistry at the electron-microscopic level. Thin sections of Araldite-embedded pituitaries from postnatal mice ranging in age from 2 days to 3–4 months were exposed to antigrowth hormone serum (anti-GH) or to anti-prolactin serum (anti-PRL) and were processed immunocytochemically by means of the colloidal-gold technique. In the normal adult pars distalis, somatotrophs reacted strongly with antiGH, and mammotrophs showed a positive anti-PRL reaction. In the dwarf adult, the granulated cells were unreactive with either anti-GH or anti-PRL. However, at 6 days postnatally, some cells in the dwarf pars distalis showed a positive reaction with anti-GH, though the reaction was not as strong as in the 6-day normal pars distalis. The 6-day dwarf was only faintly reactive with anti-PRL, and by 14 days the dwarf was unreactive with anti-GH as well as anti-PRL, in contrast to the strong positive reactivity to these two antisera in the normal 14-day pars distalis.Supported by USPHS grant HD12308     

Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands

Cannabinoids, endocannabinoids and marijuana activate two well-characterized cannabinoid receptors (CB-Rs), CB1-Rs and CB2-Rs. The expression of CB1-Rs in the brain and periphery has been well studied, but neuronal CB2-Rs have received much less attention than CB1-Rs. Many studies have now identified and characterized functional glial and neuronal CB2-Rs in the central nervous system. However, many features of CB2-R gene structure, regulation and variation remain poorly characterized in comparison with the CB1-R. In this study, we report on the discovery of a novel human CB2 gene promoter transcribing testis (CB2A) isoform with starting exon located ca 45 kb upstream from the previously identified promoter transcribing the spleen isoform (CB2B). The 5' exons of both CB2 isoforms are untranslated 5'UTRs and alternatively spliced to the major protein coding exon of the CB2 gene. CB2A is expressed higher in testis and brain than CB2B that is expressed higher in other peripheral tissues than CB2A. Species comparison found that the CB2 gene of human, rat and mouse genomes deviated in their gene structures and isoform expression patterns. mCB2A expression was increased significantly in the cerebellum of mice treated with the CB-R mixed agonist, WIN55212-2. These results provide much improved information about CB2 gene structure and its human and rodent variants that should be considered in developing CB2-R-based therapeutic agents.     

Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB(1) and CB(2) cannabinoid receptors in regulating CNS autoimmunity. We found that CB(1) receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB(2) receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB(2)-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB(2) receptor.     

The inhibitory action of exo- and endocannabinoids on [³H]GABA release are mediated by both CB₁and CB₂receptors in the mouse hippocampus

Exogenous and endogenous cannabinoids play an important role in modulating the release of neurotransmitters in hippocampal excitatory and inhibitory networks, thus having profound effect on higher cognitive and emotional functions such as learning and memory. In this study we have studied the effect of cannabinoid agonists on the potassium depolarization-evoked [(3)H]GABA release from hippocampal synaptosomes in the wild-type (WT) and cannabinoid 1 receptor (CB(1)R)-null mutant mice. All tested cannabinoid agonists (WIN55,212-2, CP55,940, HU-210, 2-arachidonoyl-glycerol, 2-AG; delta-9-tetra-hydrocannabinol, THC) inhibited [(3)H]GABA release in WT mice with the following rank order of agonist potency: HU-210>CP55,490>WIN55,212-2>2-AG>THC. By contrast, 2-AG and THC displayed the greatest efficacy eliciting almost complete inhibition of evoked [(3)H]GABA efflux, whereas the maximal inhibition obtained by HU-210, CP55,490, and WIN55,212-2 were less, eliciting not more than 40% inhibition. The inhibitory effect of WIN55,212-2, THC and 2-AG on evoked [(3)H]GABA efflux was antagonized by the CB(1) receptor inverse agonist AM251 (0.5 μM) in the WT mice. In the CB(1)R knockout mice the inhibitory effects of all three agonists were attenuated. In these mice, AM251 did not antagonize, but further reduced the [(3)H]GABA release in the presence of the synthetic agonist WIN55,212-2. By contrast, the concentration-dependent inhibitory effects of THC and 2-AG were partially antagonized by AM251 in the absence of CB(1) receptors. Finally, the inhibition of evoked [(3)H]GABA efflux by THC and 2-AG was also partially attenuated by AM630 (1 μM), the CB(2) receptor-selective antagonist, both in WT and CB(1) knockout mice. Our data prove the involvement of CB(1) receptors in the effect of exo- and endocannabinoids on GABA efflux from hippocampal nerve terminals. In addition, in the effect of the exocannabinoid THC and the endocannabinoid 2-AG, non-CB(1), probably CB(2)-like receptors are also involved.