A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

Sstr2A: a relevant target for the delivery of genes into human glioblastoma cells using fiber-modified adenoviral vectors.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

The smallest active carbamoyl phosphate synthetase was identified in the human gut archaeon Methanobrevibacter smithii.

The genome of the major intestinal archaeon Methanobrevibacter smithii contains a complex gene system coding for carbamoyl phosphate synthetase (CPSase) composed of both full-length and reduced-size synthetase subunits. These ammonia-metabolizing enzymes could play a key role in controlling ammonia assimilation in M. smithii, affecting the metabolism of gut bacterial microbiota, with an impact on host obesity. In this study, we isolated and characterized the small (41 kDa) CPSase homolog from M. smithii. The gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme was purified in one step. Chemical cross-linking and size exclusion chromatography indicated a homodimeric/tetrameric structure, in accordance with a dimer-based CPSase activity and reaction mechanism. This small enzyme, MS-s, synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia and catalyzed the same ATP-dependent partial reactions observed for full-length CPSases. Steady-state kinetics revealed a high apparent affinity for ATP and ammonia. Sequence comparisons, molecular modeling, and kinetic studies suggest that this enzyme corresponds to one of the two synthetase domains of the full-length CPSase that catalyze the ATP-dependent phosphorylations involved in the three-step synthesis of carbamoyl phosphate. This protein represents the smallest naturally occurring active CPSase characterized thus far. The small M. smithii CPSase appears to be specialized for carbamoyl phosphate metabolism in methanogens.

Lesion of the amygdala on the right and left side suppresses testosterone secretion but only left-sided intervention decreases serum luteinizing hormone level.

The effect of right- and left-sided intra-amygdaloid injection of kainic acid on the hypothalamo-hypophyseal-testicular axis was studied in rats. Both right- and left-sided injection of the neurotoxin into the amygdala resulted in a significant decrease in basal testosterone secretion in vitro of both testes and in serum testosterone concentration. In addition, left-sided administration of kainic acid significantly suppressed serum luteinizing hormone level, while right-sided intervention did not alter this parameter. The results of the present study provide further evidence on the involvement of the amygdala in the control of testicular steroidogenesis. Furthermore, the observations suggest functional asymmetry of the amygdala concerning the mechanism of suppressed testosterone secretion.

Comparison of somatostatin receptor expression in human gliomas and medulloblastomas.

The expression of the five somatostatin receptor subtypes, sst1-5 was compared on tissue containing glial tumours (glioblastomas or oligodendrogliomas), medulloblastomas, and on normal human cortex. By semiquantitative reverse transcription coupled to polymerase chain reaction, the receptor expression profiles were high in cortex and in tissue containing oligodendrogliomas. It was moderate in medulloblastomas. Tissue containing glioblastomas displayed lower expression of somatostatin receptor subtypes, sst1 and sst3 being mostly expressed. By 125I-Tyr0DTrp8 somatostatin-14 or 125I-Leu8DTrp22 Tyr25 somatostatin-28 autoradiography combined with synaptophysin immunohistochemistry, it was possible to differentiate between isolated tumoral cell component infiltrating the cerebral parenchyma (cortex or white matter) and tumoral tissue (without residual parenchyma) in glioblastomas or oligodendrogliomas. Glial tumoral tissue per se presented few somatostatin receptors. By contrast, medulloblastoma tumoral cells exhibited numerous octreotide sensitive somatostatin receptors. sst2 immunocytochemistry demonstrated immunostaining of neuronal cells and neuropile; sst2 and sst3 immunostaining was identified on glioblastoma proliferating vessels endothelial cells and on medulloblastomas tumoral cells. Faint sst2 immunostaining among glial tumoral cells was due to microglia, while glioma cells did not significantly stain. In summary, medulloblastoma tumoral cells express sst2/sst3 receptors at a high level while glioma cells do not. In gliomas, sst expression is restricted to endothelial cells on proliferating vessels (displaying both sst2 and sst3 receptors), including parenchyma and reactive microglia (only sst2). The differential expression of sst2/sst3 receptors on gliomas and medulloblastomas has implications for the therapy of these tumours.

Lesion of the insular cortex affects luteinizing hormone and testosterone secretion of rat. Lateralized effect.

The possible involvement of the insular cortex in the neural control of the hypophyseal-testicular axis was studied in male rats. Right- but not left-sided lesion of the insular cortex resulted in a significant decrease in basal testosterone secretion in vitro and serum testosterone concentration. Both right- and left-sided lesions of the insular cortex induced significant increase in serum luteinizing hormone (LH) concentration. Unilateral lesion of the insular cortex on either sides had no effect on serum follicle stimulating hormone (FSH) level. The results indicate that the insular cortex is involved in the control of testosterone and LH secretion. The data further suggest that the right insular cortex plays a predominant role in the control of male endocrine reproductive processes.

Cellular biology of somatostatin receptors.

Somatostatin, and the recently discovered neuropeptide cortistatin, exert their physiological actions via a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, sst5). Following the cloning of somatostatin receptors significant advances have been made in our understanding of their molecular, pharmacological and signaling properties although much progress remains to be done to define their physiological role in vivo. In this review, the present knowledge regarding neuroanatomical localization, signal transduction pathways, desensitization and internalization properties of somatostatin receptors is summarized. Evidence that somatostatin receptors can form homo- and heterodimers and can physically interact with members of the SSTRIP/Shank/ProSAP1/CortBP1 family is also discussed.

In vivo internalization of the somatostatin sst2A receptor in rat brain: evidence for translocation of cell-surface receptors into the endosomal recycling pathway.

To determine whether cellular compartmentalization of somatostatin receptors can be regulated in vivo, we examined the immunocytochemical distribution of the sst2A receptor (sst2AR) after stereotaxical injections of somatostatin analogs into the rat parietal cortex. Whereas CH-275, a sst1R agonist, failed to induce changes in the diffuse sst2AR immunostaining pattern characteristic of control animals, somatodendritic profiles displaying intracytoplasmic immunoreactive granules became apparent short-term after injection of either somatostatin or the sst2R agonist octreotide. Confocal microscopy revealed that 90% of sst2AR-immunoreactive endosome-like organelles displayed transferrin receptor immunoreactivity. At the electron microscopic level, the percentage of sst2AR immunoparticles dramatically decreased at the plasmalemma of perikarya and dendrites after octreotide injection. Conversely, it significantly increased in endosomes-like organelles. These results demonstrate that sst2ARs undergo, in vivo, rapid and massive internalization into the endocytic recycling compartment in response to acute agonist stimulation and provide important clues toward elucidating somatostatin receptor signaling in the mammalian brain.

The effect of callosotomy on testicular steroidogenesis in hemiorchidectomized rats: a pituitary-independent regulatory mechanism.

In recent years, increasing number of data indicate that cerebral structures exert a direct, pituitary-independent, neural regulatory action on the endocrine glands. In addition, both experimental and clinical observations indicate functional asymmetry of the control system. Therefore, the objective of the present study was to study the effect of callosotomy on testicular steroidogenesis and serum gonadotrop concentrations in rats subjected to left- or right-sided orchidectomy. In animals underwent callosotomy plus left-sided orchidectomy the basal testosterone secretion in vitro of the remaining (right) testis was significantly higher than that of intact controls, and of rats subjected to sham surgery plus left orchidectomy. In contrast, either sham operation or callosotomy plus right-sided orchidectomy did not interfere with testicular steroidogenesis. Sham surgery or callosotomy plus left orchidectomy induced a significant rise in serum follicle-stimulating hormone concentration while right orchidectomy combined either with sham surgery or callosotomy did not alter this parameter. There was no statistically significant difference between experimental groups in serum testosterone and luteinizing hormone concentrations. The results indicate the involvement of the corpus callosum in a pituitary-independent neural control of testicular steroidogenesis. The data further suggest a different response in steroidogenesis of the left and the right testis following hemicastration and callosotomy.

Effect of intratesticular administration of TRH or anti-TRH antiserum on function of rat testis.

The effect of intratesticular administration of thyrotropin-releasing hormone (TRH) and anti-TRH antiserum on steroidogenesis was studied in immature and adult rats. In 9-day-old animals local administration of the neuropeptide resulted in an increase in basal testosterone secretion in vitro. Similar treatment of 15-day-old rats suppressed hCG-stimulated testosterone secretion with no change in basal testosterone production. In both immature groups the treatment did not affect serum testosterone concentration. By contrast, in adults TRH decreased serum testosterone level, but did not influence basal and hCG-stimulated testosterone secretion. Both in immature and adult rats, the changes in steroidogenesis were evident 1 hour posttreatment. Five days after the administration of anti-TRH antiserum into the remaining testis of immature rats subjected to hemicastration just prior to the antiserum treatment, the alterations in steroidogenesis were opposite to those detected after treatment with TRH. In 9-day-old rats the antiserum suppressed steroidogenesis, while in 15-day-old animals it stimulated testosterone secretion. The results suggest that testicular TRH might exert a local action on testicular steroidogenesis, and the effect is age-dependent.

Involvement of the Sst1 somatostatin receptor subtype in the intrahypothalamic neuronal network regulating growth hormone secretion: an in vitro and in vivo antisense study.

Five somatostatin (SRIH) receptors (sst1-5) have been cloned. Recent anatomical evidence suggests that sst1 and sst2 may be involved in the central regulation of GH secretion. Given the lack of specific receptor antagonists, we used selective antisense oligodeoxynucleotides (ODNs) to test the hypothesis that one or both of these subtypes are involved in the intrahypothalamic network regulating pulsatile GH secretion. In mouse neuronal hypothalamic cultures the proportion of GHRH neurons coexpressing sst1 or sst2 messenger RNAs (mRNAs) was identical. In contrast, sst1 mRNAs were more often present than sst2 in SRIH-expressing neurons. Firstly, sst1 antisense ODN in vitro treatment abolished sst1, but not sst2, receptor modulation of glutamate sensitivity and decreased sst1, but not sst2, mRNAs. The reverse was true after treatment with sst2 antisense. Sense ODNs did not alter the effects of SRIH agonists. In a second series of experiments, nonanaesthetized adult male rats were infused for 120 h intracerebroventricularly with ODNs. Only the sst1 antisense ODN diminished the amplitude of ultradian GH pulses without modifying their frequency. In parallel, sst1 antisense ODN strongly diminished sst1 immunoreactivity in the anterior periventricular nucleus and median eminence, as well as sstl periventricular nucleus mRNA levels. The effectiveness of the sst2 antisense ODN was attested by the inhibition of hypothalamic binding of [125I]Tyr0-D-Trp8-SRIH. Scrambled ODNs had no effect on GH secretion or on sst mRNAs or SRIH binding levels. These results favor a preferential involvement of sst1 receptors in the intrahypothalamic regulation of GH secretion by SRIH.

Intratesticular serotonin affects steroidogenesis in the rat testis.

The effect of intratesticular administration of serotonin (5-HT), ketanserin (5-HT2 receptor antagonist), and 5,7-dihydroxytryptamine (5,7-DHT) (the neurotoxin that destroys serotoninergic neural elements) on steroidogenesis was studied in immature and adult rats. In adults, bilateral intratesticular injection of 5-HT resulted in a significant decrease in basal but not in hCG-stimulated testosterone secretion and in serum testosterone concentration, whereas ketanserin induced a significant rise in steroidogenesis 1 h post-treatment. There was no effect 1 day after administration of 5-HT or ketanserin, and 7 days after the injection of 5,7-DHT. In immature rats 1 day after bilateral testicular administration of ketanserin, basal testosterone secretion in vitro was significantly suppressed. In immature hemicastrates, local injection of 5-HT resulted (1 day post-treatment) in a significant rise in steroidogenesis while administration of 5,7-DHT decreased testosterone secretion 7 days after the injection of the neurotoxin. The results indicate that in adult rats 5-HT exerts a suppressive, whereas in immature rats, a stimulatory action on steroidogenesis occurs. Data also suggest that, in both age groups, the effect of 5-HT is mediated through 5-HT2 receptors. The observation that in immatures administration of the neurotoxin resulted in an effect similar to that found following the treatment with the receptor antagonist suggests that, in this age group, 5-HT derived from local neural elements might also be involved in the control of 5-HT on Leydig cell steroidogenesis.

Interrelations between hypothalamic somatostatin and proopiomelanocortin neurons.

Somatostatin receptors were visualized by [125I]-Tyr0-DTrp8-somatostatin radioautography on 35% of arcuate neurons containing proopiomelanocortin (POMC) mRNA, as identified by in situ hybridization using a [35S] labelled riboprobe on 5 microm-thick consecutive sections. Furthermore, double immunohistochemical staining revealed contacts of beta-endorphin or alpha-MSH containing fibres with a majority of somatostatin perikarya in the anterior hypothalamic periventricular nucleus. Taken together, these data indicate that hypothalamic somatostatin and POMC neurons are interconnected. The results are discussed in term of intrahypothalamic control of GH secretion.

Distribution and pharmacological characterization of somatostatin receptor binding sites in the sheep brain.

Somatostatin binding sites have been localized and quantified in the sheep brain using 125I-Tyr0-DTrp8-somatostatin, by quantitative high resolution light microscopic autoradiography. Sections were analyzed by densitometry on radioautographic film, and subsequently on slides coated with photoemulsion. Specific somatostatin binding sites were concentrated in the medial habenula, superior colliculus, dorsal motor nucleus of the vagus nerve, inferior olive, spinal trigeminal nucleus, and cerebellum. In competition experiments, octreotide, a sst2/sst3/sst5 selective agonist only partially displaced 125I-Tyr0-DTrp8-somatostatin in the three cerebellar layers while it was fully active as compared to somatostatin 14 and 28 in the deeper layers of the parietal cortex. Moderate to low somatostatin receptor densities were present in the mesencephalic periaqueductal gray, dorsal raphe, thalamic paraventricular nucleus, interpeduncular nucleus, pineal gland, dorsal tegmental, dorsolateral tegmental and parabrachial nuclei, nucleus of the solitary tract. The distribution of somatostatin binding sites generally correlates with the data obtained on slides dipped in photoemulsion which provided better resolution and more precise localization. In most of the labeled areas, 125I-Tyr0-DTrp8-somatostatin receptor binding was distributed between both neuropil and perikarya. Perikarya bearing 125I-Tyr0-DTrp8-somatostatin receptors were observed in areas which did not display detectable binding sites on film such as the preoptic-anterior hypothalamic complex and arcuate nucleus and in the locus coeruleus. In conclusion, the distribution of 125I-Tyr0-DTrp8-somatostatin binding sites in sheep brain is very reminiscent of other mammals being closer to the human than to rodents.

Local effect of PACAP and VIP on testicular function in immature and adult rats.

PACAP, VIP, anti-PACAP and anti-VIP antisera were injected intratesticularly. In 9-day-old hemicastrated rats PACAP or VIP decreased basal testosterone secretion. In 22-day-old hemicastrates VIP but not PACAP reduced compensatory testicular hypertrophy, however, neither PACAP nor VIP altered steroidogenesis. Anti-VIP antiserum to this age group increased testosterone production and enhanced compensatory testicular hypertrophy. In adult hemicastrates neither the peptides nor the antisera influenced steroidogenesis. Neither in immatures nor in adults treatment of both testes with PACAP or VIP had any effect. Data indicate that both PACAP and VIP might exert a local action on testicular steroidogenesis, on compensatory testicular hypertrophy, and these effects are age-dependent.

Testicular injection of 5,6-dihydroxytryptamine or vasectomy interferes with the local stimulatory effect of oxytocin on testicular steroidogenesis in immature rats.

Previous studies indicated that in immature rats testicular administration of oxytocin stimulates testicular steroidogenesis. In the present study, testicular treatment with oxytocin (50 ng) was combined with pharmacological or surgical denervation of the testis in hemigonadectomized immature rats. For denervation 5,6-dihydroxytryptamine (160 micrograms/testis), a substance that destroys serotoninergic neuronal elements, was injected intratesticularly or vasectomy was performed, which also includes transection of the inferior testicular nerve. In 9-day-old animals both vasectomy and pretreatment of the testis with 5,6-dihydroxytryptamine prevented the oxytocin-induced rise in serum testosterone concentration. In addition, intratesticular injection of oxytocin combined with vasectomy resulted in a significant increase in in vitro basal testosterone secretion of the testis. A similar effect was not observed in the 5,6-dihydroxytryptamine-pretreated group receiving oxytocin. The results indicate that testicular innervation is involved in the control of local peptide effects, and data further suggest a differential role of these neural elements in intratesticular regulatory processes.

Effect of intratesticular administration of somatostatin on testicular function in immature and adult rats.

Somatostatin has been demonstrated in the testis. In the present investigations the effect of intratesticular injection of somatostatin on serum testosterone level and in vitro basal testosterone secretion of the testis was studied in immature and adult rats. Intratesticular injection of somatostatin in adult rats with two testes in situ decreased serum testosterone concentration and basal testosterone secretion in vitro. Similar treatment in immature animals had no effect on the parameters studied. In immature hemicastrates, however, local administration of the peptide induced a significant rise both in basal testosterone secretion in vitro and serum testosterone level, and resulted in an increase in testicular weight. In adult hemicastrates the peptide did not influence testicular functions. These results indicate that somatostatin might play a modulatory role in testicular steroidogenesis. The data also suggest that the effect of somatostatin is age-dependent, and that hemicastration might modify local action of the peptide.

Involvement of a direct neural mechanism in the control of gonadal functions.

Much time has been devoted to study of the hypothalamo-hypophyseal-gonadal axis. However, there is now evidence of a complementary control mechanism for the gonads, namely a pituitary-independent, direct neural link that exists between the central nervous system and the gonads. We investigated whether mediobasal temporal lobe structures could control gonadal functions by a purely neural mechanism or whether they acted through the classical hypothalamo-hypophyseal system. Right- or left-sided deafferentiation of the temporal lobe was combined with right- or left-sided hemicastration in adult and prepubertal male and female rats. In adult females right-sided deafferentiation, regardless of the side of hemiovariectomy significantly reduced the extent of compensatory ovarian hypertrophy. Similar lesions on the left side did not interfere with the usual compensatory ovarian growth. This difference in compensatory hypertrophy between right- and left-sided lesioned rats was observed even in the face of a significant drop in serum LH concentrations in both groups. In pre- and postpubertal females temporal lobe lesion in either side was unable to alter compensatory hypertrophy or serum LH or progesterone concentrations. In adult male rats only left-sided deafferentiation combined with left orchidectomy resulted in decreased T production, while in prepubertal male rats, only right-sided brain surgery plus left orchidectomy resulted in a significant decrease in basal testosterone secretion of the remaining testis. These findings indicate that mediobasal temporolimbic structures are involved in the neural control of gonadal functions. It appears that this lateralized mechanism is age- and sex-dependent.

Growth hormone-releasing hormone, somatostatin, galanin and beta-endorphin afferents to the hypothalamic periventricular nucleus.

A combined retrograde tracing (wheat germ agglutinin-horseradish peroxidase-gold complex)-immunohistochemical technique was used to identify the origin of growth hormone-releasing hormone (GHRH)-immunoreactive (ir), beta-endorphin-ir, galanin (GAL)-ir and somatostatin (SRIH)-ir terminals in the hypothalamic periventricular nucleus, which contains all the hypophysiotrophic SRIH-ir neurons. Retrogradely labeled cells were mostly observed ipsilaterally in the arcuate, dorsomedial (DMH), suprachiasmatic nuclei and the parvocellular part of the paraventricular nucleus. They were less abundant in the ventromedial and periventricular nuclei and in the lateral hypothalamus. The proportion of retrogradely labeled GHRH cells was greater at the outer rim of the ventromedial nucleus (10%) than in the arcuate nucleus proper (3%). In the arcuate nucleus, 14% of the SRIH-ir cells projected to the periventricular nucleus. Of the GAL-ir cells in the arcuate and the DMH 10% were double-labeled. Scattered retrogradely labeled GAL-ir cells were observed in paraventricular and perifornical nuclei and in the lateral hypothalamus. Of the beta-Endorphin-ir cells in the ventral part of the arcuate nucleus 15% were retrogradely labeled. It is concluded that: (1) There is no major direct connection between the hypophysiotropic GHRH and SRIH neurons, respectively, located in the arcuate and periventricular nucleus. (2) GHRH projections to the periventricular nucleus arise mainly from cells located at the outer rim of the ventromedial nucleus. (3) Intrahypothalamic SRIH projections to the periventricular nucleus arise from arcuate SRIH neurons located along the wall of the third ventricle. (4) GAL neurons from the DMH and the arcuate nucleus innervate to the same extent the periventricular nucleus. (5) beta-Endorphin arcuate neurons strongly innervate the periventricular nucleus.

Effect of unilateral deafferentiation in the medial basal portion of the temporal lobe on the hypophyseo-ovarian axis in rats: an age-dependent lateralized control mechanism.

The possible physiological role of the medial basal portion of the temporal lobe (including the corticomedial amygdaloid nucleus) in the neural control of the hypophyseo-ovarian axis was studied in pre- and postpubertal as well as in adult rats. Unilateral deafferentiation of a small medio-basal portion of the temporal lobe was performed in unilaterally ovariectomized animals, and the rate of compensatory hypertrophy of the remaining ovary was recorded. In adults compensatory ovarian hypertrophy was significantly reduced following right- but not left-sided deafferentiation. Temporal lobe surgery did not significantly influence the usual compensatory ovarian growth in pre- and postpubertal rats. Serum luteinizing hormone levels decreased significantly in adults regardless of the side of brain interventions, while no obvious change in the hormone concentration could be observed in prepubertals. Serum follicle-stimulating hormone concentrations showed no alterations in any experimental group. In postpubertal rats the serum progesterone level was unchanged following brain surgery. The present observations indicate that unilateral deafferentation in the temporal lobe could modify compensatory ovarian hypertrophy by a direct neural mechanism and data further suggest functional laterality of these structures in the control of ovarian functions.