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BACKGROUND The gastrointestinal (GI) barrier can be damaged by chemotherapy or radiation therapy, causing fatigue, malnutrition, sepsis, dose-limiting toxicity, and, occasionally, death. Glucagon-like peptide-2 (GLP-2) promotes mucosal epithelium growth and repair in the GI tract. Here, we examined the GI-protective effects of apraglutide, a long-acting peptide GLP-2 analog, in murine models of chemotherapy, and total body irradiation followed by allogeneic transplantation. MATERIAL AND METHODS The impact of apraglutide on cytarabine or melphalan chemotherapy-induced intestinal damage was assessed in BALB/c mice, and the effect on allogeneic transplantation in BALB/cJ and C57BL/6J mice. Outcomes included survival, and changes in body weight, intestinal function and morphology, including colon length and bacterial composition of the intestinal microbiota. RESULTS Adding apraglutide to chemotherapy significantly improved survival rates and reduced weight loss, with no impact on leukocyte counts (and, therefore, no effect on chemotherapy-induced immunosuppression), compared with chemotherapy alone in mice. These benefits were associated with preservation of the morphological integrity of the GI mucosa, attenuation of the negative impact of cytarabine on the intestinal microbiota, and significant improvement in plasma levels of citrulline. In addition, in a model of irradiation followed by allogeneic transplantation, mice in groups receiving apraglutide had improved survival, reduced weight loss, and increased colon length compared with those that did not. CONCLUSIONS Apraglutide protects intestinal function and improves survival in mice following allogeneic transplantation or chemotherapy with cytarabine or melphalan. The potential effect of apraglutide on chemotherapy efficacy and on engraftment following allogeneic transplantation has been investigated in a parallel manuscript.

Background: Short-bowel syndrome is the leading cause of pediatric intestinal failure, resulting in dependency on long-term parenteral nutrition (PN). To promote enteral autonomy in neonates, a key outcome may be intestinal growth in length. The purpose of this study was to determine if intestinal lengthening persists following discontinuation of treatment with 1 of 2 GLP-2 analogues with different pharmacokinetic profiles., Methods: Neonatal short-bowel piglets were assigned to saline control (S), 7-day treatment with teduglutide (T) (0.05 mg/kg twice daily), or 7-day treatment with apraglutide (A) (5 mg/kg twice weekly). Comparisons were made between day 7 and day 14 endpoints using analysis of variance. Data included small-intestine length, weight, histology, and quantitative polymerase chain reaction analysis of mucosal transcripts for peptide growth factors and their receptors, nutrient transporters, and tight-junction proteins., Results: Compared with control, 7 days of GLP-2 analogue treatment induced mucosal adaptation based on villus hyperplasia (P = .003), which was not durable 7 days after treatment cessation (day 14; P = .081). Treatment increased intestinal growth in length by day 7 (P = .005), which was maintained (by T) or further increased (by A) at day 14 (P < .001). No significant differences in mucosal transcripts were detected., Conclusion: Unlike mucosal adaptation, intestinal growth appears to be a lasting outcome of treatment with long-acting GLP-2 analogues in a neonatal piglet short-bowel model. This has significant clinical implications for neonates, given their potential for intestinal growth. Intestinal lengthening varies between analogues with different half-lives; however, molecular mechanisms require further elucidation., (© 2020 American Society for Parenteral and Enteral Nutrition.)

Background: In treating short-bowel syndrome (SBS), autonomy from parenteral nutrition (PN) relies upon intestinal adaptation, which can be augmented by glucagon-like peptide-2 (GLP-2) analogues. In neonatal piglets with SBS, we compared intestinal adaptation following treatment with 2 GLP-2 analogues: teduglutide (TED) and apraglutide (APRA) METHODS: Following 75% distal small-intestinal resection, piglets were allocated to 4 treatment groups: saline (CON: n = 8), twice weekly APRA (5 mg/kg/dose; n = 8), and TED once daily (TED, 0.05 mg/kg/dose; n = 8) or twice daily (TEDBID, 0.05 mg/kg/dose; n = 7). Pharmacokinetic (PK) studies were undertaken, and on day 7, small-intestinal length and weight were measured and jejunal tissue collected for histology., Results: PK profiles were different between the 2 analogues. To achieve a comparable exposure to APRA, TED requires twice daily injection (TEDBID). Compared with CON, APRA and TEDBID increased small-bowel length (cm) (CON: 141, APRA: 166, TED: 153, TEDBID: 165; P = .004), whereas APRA increased small-bowel weight (g) (CON: 26, APRA: 33, TED: 28, TEDBID: 31; P = .007) and villus height (mm) (CON: 0.59, APRA: 0.90, TED: 0.58, TEDBID: 0.74; P < .001)., Conclusion: APRA injected only twice during the 7 consecutive days demonstrated a superior intestinotrophic effect compared with TED injected once daily. Even at more comparable drug exposure, when TED was injected twice a day, APRA showed superior trophic activity at the mucosal level. This is highly relevant for the treatment of pediatric SBS, given the markedly lower dose frequency by subcutaneous injection of APRA., (© 2020 American Society for Parenteral and Enteral Nutrition.)

Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33-amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS. SIGNIFICANCE STATEMENT: Apraglutide is a potent and selective GLP-2 agonist with an extremely low clearance and prolonged elimination half-life, which differentiates it from teduglutide (the only approved GLP-2 agonist). The enhanced pharmacokinetics of apraglutide will benefit patients by enabling a reduced dosing frequency and removing the need for daily injections., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Both the antiangiogenic and antitumoral activity of shark cartilage extracts (SCE) have been demonstrated in animal models and clinical trials. Studies reported that SCE induces the expression of tissue plasminogen activator gene (PLAT) in endothelial cells and increases the activity of the protein (t-PA) in vitro. The aim of this study was to demonstrate the crucial role of t-PA induction in the antiangiogenic and antitumor activity of SCE in experimental glioma. This study showed antiangiogenic and antitumoral effects of SCE in three mice glioma models (C6, HGD and GL26). Histological examination suggested perivascular proteolysis and edema as well as important intratumoral necrosis, which artefactually increased the tumor volume at high doses. Thus, the antiangiogenic effect of SCE correlated with the presence of t-PA and angiostatin in degenerating vessels. Functional in vivo experiments were conducted to modulate the plasminogen pathway. No antiangiogenic effect was observed on tumors overexpressing the plasminogen activator inhibitor-1 (PAI-1). Moreover, therapeutical effects were neutralized in mice that were cotreated with ε-aminocaproic acid (EACA, 120 mg/kg p.o.), an inhibitor that blocks the high-affinity lysine binding sites of both plasminogen and plasmin. In contrast, cotreatment with N-acetylcysteine (NAC, 7,5mg/kg i.p.), a sulfhydril donor that reduces plasmin into angiostatin or other antiangiogenic fragments, increased the benefit of SCE on mice survival. In subcutaneous models, NAC prevented the increase in tumor volume caused by high doses of cartilage extract. In conclusion, this study indicates that induction of t-PA by shark cartilage extract plays an essential role in its antiangiogenic activity, but that control of excessive proteolysis by a plasmin reductor could prevent edema and uncover the full benefit of shark cartilage extract in the treatment of intracranial tumors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, which exerts direct effects on vascular endothelial cells, including endothelial cell proliferation and survival, tubulogenesis, and vascular permeability. In this study, we examined whether Neovastat, a naturally occurring multifunctional antiangiogenic drug, could inhibit the endothelial cell response to VEGF stimulation., Results: We demonstrated that Neovastat was able to block the VEGF-dependent microvessel sprouting from Matrigel-embedded rat aortic rings, and it also blocked the VEGF-induced endothelial cell tubulogenesis in vitro. In vivo studies showed that Neovastat was able to specifically inhibit VEGF-induced plasma extravasation in numerous tissues, including pancreas and skin. The mechanism of action of Neovastat on VEGF-mediated effects was also evaluated at the molecular level. Neovastat was shown to compete against the binding of VEGF to its receptor in endothelial cells and significantly inhibited the VEGF-dependent tyrosine phosphorylation of VEGF receptor-2, whereas it had no significant effect on VEGF receptor-1 activity. Moreover, the inhibition of receptor phosphorylation was correlated with a marked decrease in the ability of VEGF to induce pERK activation. Neovastat does not compete against the binding of basic fibroblast growth factor, indicating a preferential inhibitory effect on the VEGF receptor., Conclusions: Because Neovastat was shown previously to inhibit metalloproteinase activities, these results suggest that Neovastat is able to target multiple steps in tumor neovascularization, further emphasizing its use as a pleiotropic, multifunctional antiangiogenic drug.

A novel naturally occurring antiangiogenic agent isolated from cartilage, referred to as Neovastat (AE-941), was examined for its efficacy against tumor neovascularization and progression. Exposure to Neovastat results in ex ovo antiangiogenic properties in the chorioallantoid membrane of chicken embryo (71% decrease in the angiogenic index as compared to the basic fibroblast growth factor (bFGF) treated control embryos, P < 0.0001). Oral administration of Neovastat inhibits bFGF-induced angiogenesis in the Matrigel mouse model (87.5% decrease in hemoglobin as compared to the bFGF-treated control implants, P < 0.0001). Neovastat also induces a dose response decrease of lung metastases in the Lewis lung carcinoma model (oral administration; 69.1% of inhibition obtained at the maximal dose of 0.5 ml/day, P < 0.0001). Combined with a sub-optimal dose of cisplatinum (2 mg/kg, i.p.), Neovastat (0.5 ml/day) improved the therapeutic index by increasing the antimetastatic efficacy and by exerting a protective activity against cisplatinum-induced body weight loss and myelosuppression. In summary, our experimental data provide evidence of antiangiogenic and antimetastatic properties of Neovastat, following oral administration.

Experimental allergic encephalomyelitis (EAE) has been used as an animal model for the human demyelinating disease multiple sclerosis (MS). In acute MS or EAE, early disruption in the integrity of the blood-brain-barrier (BBB) precedes brain infiltration by inflammatory cells or any clinical evidence of disease. BBB permeability could be affected by vasoactive mediators and cytokines released from perivascular brain mast cells. We investigated the number and degree of activation of brain mast cells in EAE and the effect of the heterocyclic histamine-1 receptor antagonist hydroxyzine, a piperazine compound known to also block mast cells. Acute EAE was induced in Lewis rats by immunization with whole guinea pig spinal cord homogenate and complete Freund's adjuvant (CFA). A second group of animals were treated orally with hydroxyzine for one day before immunization and then continuously for 14 days. Control rats were treated with CFA or hydroxyzine alone. The clinical progression of EAE was assessed on days 10, 12 and 14 after immunization. The number of metachromatic mast cells and the degree of degranulation was assessed in the thalamus with light microscopy. At day 14, there was a three-fold increase in the number of brain mast cells with EAE, as compared to controls. These cells were positive for the immunoglobulin E binding protein (FcepsilonRI), while those from control rats were not. Over 40% of all thalamic mast cells studied in EAE showed partial staining or extruded secretory granule indicative of secretion. Hydroxyzine treatment inhibited (p<0.05) the progression and severity of EAE by 50% and the extent of mast cell degranulation by 70% (p<0.05). These findings indicate that brain mast cells are associated with EAE development and that inhibition of their activation correlates positively with the clinical outcome.

Mast cells hold a key position in the defensive mechanisms against exogenous intruders. In this study, we investigated whether human mast cells express functional major histocompatibility complex (MHC) class II molecules that can transduce endogenous signals and present staphylococcal enterotoxin A (SEA) to T cells. Similar to HMC-1 human mast cell line, umbilical cord blood-derived mast cells express HLA-DR, -DP and -DQ molecules on their surface. MHC class II molecules expressed on HMC-1 cells bind significantly the SEA (a natural MHC class II ligand), and their ligation with specific mAbs or with SEA, leads ultrastructural changes, suggesting their degranulation. Recognition of SEA-bound MHC class II molecules on HMC-1 mast cells by the T cell receptor of K25 cells, an SEA-specific murine T cell hybridoma, triggers significant IL-2 secretion by these T cell hybridomas. Hence, our data point out the expression of functional MHC class II molecules on human mast cells, reinforcing the implication of these cells in the defense mechanisms of acquired immunity.

Neuromuscular transmission and muscle activity during early stages of embryonic development are known to influence the differentiation and survival of motoneurons and to affect interactions with their muscle targets. We have examined neuromuscular development in an avian genetic mutant, crooked neck dwarf (cn/cn), in which a major phenotype is the chronic absence of the spontaneous, neurally mediated movements (motility) that are characteristic of avian and other vertebrate embryos and fetuses. The primary genetic defect in cn/cn embryos responsible for the absence of motility appears to be the lack of excitation-contraction coupling. Although motility in mutant embryos is absent from the onset of activity on embryonic days (E) 3-4, muscle differentiation appears histologically normal up to about E8. After E8, however, previously separate muscles fuse or coalesce secondarily, and myotubes exhibit a progressive series of histological and ultrastructural degenerative changes, including disarrayed myofibrils, dilated sarcoplasmic vesicles, nuclear membrane blebbing, mitochondrial swelling, nuclear inclusions, and absence of junctional end feet. Mutant muscle cells do not develop beyond the myotube stage, and by E18-E20 most muscles have almost completely degenerated. Prior to their breakdown and degeneration, mutant muscles are innervated and synaptic contacts are established. In fact, quantitative analysis indicates that, prior to the onset of muscle degeneration, mutant muscles are hyperinnervated. There is increased branching of motoneuron axons and an increased number of synaptic contacts in the mutant muscle on E8. Naturally occurring cell death of limb-innervating motoneurons is also significantly reduced in cn/cn embryos. Mutant embryos have 30-40% more motoneurons in the brachial and lumbar spinal cord by the end of the normal period of cell death. Electrophysiological recordings (electromyographic and direct records form muscle nerves) failed to detect any differences in the activity of control vs. mutant embryos despite the absence of muscular contractile activity in the mutant embryos. The alpha-ryanodine receptor that is genetically abnormal in homozygote cn/cn embryos is not normally expressed in the spinal cord. Taken together, these data argue against the possibility that the mutant phenotype described here is caused by the perturbation of a central nervous system (CNS)-expressed alpha-ryanodine receptor. The hyperinnervation of skeletal muscle and the reduction of motoneuron death that are observed in cn/cn embryos also occur in genetically paralyzed mouse embryos and in pharmacologically paralyzed avian and rat embryos. Because a primary common feature in all three of these models is the absence of muscle activity, it seems likely that the peripheral excitation of muscle by motoneurons during normal development is a major factor in regulating retrograde muscle-derived (or muscle-associated) signals that control motoneuron differentiation and survival.

This review comes up with the possible association between mast cells and tumour progression and summarizes some of the most recent data on the subject. The accumulation of mast cells around tumour areas is a very old observation. However, the functional significance of such phenomenon is a subject of controversy because of contradictory experimental data. In this review, two hypotheses are suggested. The first, refers on the possibility that the accumulation of mast cells is part of a general immunological host-defense reaction since, mast cells have been shown to be cytotoxic for some tumours (especially those sensitive to tumor necrosis factor-alpha). However, if such hypothesis is correct, one should explain why in most clinical and experimental cases, tumours continue to progress although the high incidence of such immune's system cells. We are therefore brought to consider a second possibility, in which, mast cells products could promote tumoural growth and metastasis. In fact, it is well documented that heparin, combined to a range of heparin-binding factors such as bFGF or TGF beta is able to promote neovascularisation, and that mast cell proteases cause cell structural alterations and loss of the extracellular matrix integrity. The role of histamine secreted by mast cells is less clear. There is indeed controversial experimental data referring to histamine's content within tumoural tissues and to histamine's proper effect on tumour expansion. Finally, this review discuss the mechanisms resulting to mast cell accumulation around tumours and more particularly the contribution of tumoural cells.

We investigated the ability of important regulators of osteoblast function, such as insulin-like growth factor I (IGF-I), transforming growth factor beta 1 (TGF beta 1), and urokinase-type plasminogen activator (uPA) to act as mediators in cell-cell interactions between osteoblast-like cells and metastatic prostate cancer cells, in vitro. In addition, we assessed whether these growth substances can (a) mediate glucocorticoid receptor (GR) function and (b) be implicated in dexamethasone-induced regression of osteoblastic tumors. Exogenous IGF-I, rat/human uPA, and PA-III (rat)/PC-3 (human) prostate cancer cells conditioned media (CM) stimulated the proliferation of rat (UMR 106 cells) and human (MG-63 cells) osteosarcoma cells. This mitogenic activity was completely neutralized by anti-IGF-I specific antibody. In addition, dexamethasone decreased cell growth, up regulated TGF beta 1 mRNA, and down regulated uPA mRNA expression in prostate cancer cells. Furthermore, it inhibited cell growth by activating latent-TGF beta 1 in osteoblast-like cells. In addition, dexamethasone down regulated the expression of IGF-I mRNA in osteoblast-like cells. Therefore, it is conceivable that uPA, TGF beta 1 and IGF-I mediate at least in part cell-cell interactions and GR function in osteoblastic metastases. Conceivably, regression of the osteoblastic tumors produced by high-dose dexamethasone treatment in hormone-refractory prostate cancer patients is been mediated by differential regulation of growth factors, locally.

The evolution of rat cerebral mast cell phenotype during development was studied using antibodies against the granule chymases, rat mast cell protease I (RMCP-I) and rat mast cell protease II (RMCP-II) and their gene transcripts, as markers for serosal and mucosal mast cells, respectively. In situ hybridization using specific oligoprobes for RMCP-II permitted visualization of RMCP-II mRNA-containing cells as early as day 15 of embryonic development (E15). From E19 to day 4 postpartum (D4) their number increased whilst they migrated from the pia mater to the choroid fissure; at D8 cells expressing RMCP-II gene transcripts were no longer observed. The 3'-end untranslated nucleotide sequence of the RMCP-I cDNA was established in order to design selective cDNA probes for Northern blot analysis of both enzymes. Northern blot analysis revealed a strong expression of RMCP-I and RMCP-II mRNAs at D2. At D4, RMCP-I mRNA expression was still high, whereas that of RMCP-II was decreased. In adult brain, mRNA expression for both proteases was low, but detectable. Quantification of both proteases by ELISA showed that, from E19 to D4, levels of RMCP-II were maximal at E19 and remained constant until D4, whereas RMCP-I increased as a function of age. Thereafter, levels of both proteases decreased progressively, but were still present in the adult brain, with RMCP-II being uniformly distributed and RMCP-I concentrated in the thalamus. Immunohistochemical staining showed RMCP-II-immunoreactive cells within the pia mater at E19; on D2 and D4, cells with both RMCP-I and RMCP-II immunoreactivities were found within the choroid fissure and from D8, only RMCP-I-immunoreactive mast cells were observed. In the thalamus of adult rats, the latter had a perivascular localization. This study shows that in the adult, both types of mast cells are present, although in small numbers, except for RMCP-I-immunoreactive mast cells which are abundant in the thalamus. The changes in the number and phenotype of cerebral mast cells may result from the influence of a number of growth factors during development.

1. Lung transplantation causes a total interruption of the inneration and vascularization within the transplanted organ, followed by repair processes. This is frequently associated with bronchial hyper-responsiveness. A common feature of tissue repair is an increase in the number of mast cells. Three phenotypically distinct mast cell subsets, with respect to their protease content, have been identified in rat lung, and it is probable that mast cells of differing protease phenotype fulfil different functions. 2. We have compared the number, protease phenotype and distribution of mast cells in left lung from transplanted and control Lewis rats 1 month after syngeneic unilateral left lung transplantation, without interference of inflammation, graft rejection or of any treatment. Connective and mucosal-type mast cell phenotypes were characterized using antibodies directed against their specific rat mast cell proteases, RMCPI and RMCPII, respectively. 3. After transplantation, RMCPI and RMCPII tissue concentrations increased by 172% and 239%, respectively, compared with controls (13.1 +/- 1.2 and 5.6 +/- 1.0 micrograms/g). 4. Localization of mast cell phenotypes was studied by immunohistochemistry after double immunostaining. The number of mast cells increased after transplantation: the increase in the number of RMCPI-immunoreactive mast cells (RMCPI+) was significant around bronchioles and arterioles, around large vessels and in the pleura. The number of RMCPII+ mast cells also significantly increased around bronchioles and arterioles, as well as in the smooth muscle layer of large airways. Some mast cells stained for the presence of both RMCPI and RMCPII, supporting the existence of co-expressing phenotype in rat lung. The number of mast cells of the RMCPI+/II+ phenotype significantly increased around bronchioles and arterioles and in the pleura. Moreover, the distribution of the mast cell phenotypes was modified in the different areas after transplantation. 5. This indicates a local differentiation/maturation of mast cells after transplantation.

Thioperamide, an H3-receptor antagonist that enhances endogenous histamine release, induced c-fos mRNA expression and Fos-like immunoreactivity in magnocellular neurones of rat supraoptic and paraventricular nuclei. This response was prevented as a result of blockade of the H1 receptor, indicating that endogenous histamine is able to activate these magnocellular neurones via stimulation of this receptor.

Transfection of a human dopamine D3 receptor cDNA in a neuroblastoma-glioma hybrid cell line (NG 108-15) provided clonal cell lines stably expressing up to 600 fmol per mg protein of [125I]iodosulpiride binding sites. Dopamine and several agonists distinguished two receptor-affinity states in membranes. In the case of dopamine, the high-affinity state (Ki = 0.9 nM, 30% of total binding) was completely converted into a low-affinity state (Ki = 57 nM) in the presence of 10 microM guanosine-5'-O-(3-thiotriphosphate). In addition to these two sites, a site with a very low affinity for dopamine was evidenced in whole cells. The dopamine D3 receptor mediated two responses: c-fos activation, as measured by the appearance of Fos-like immunoreactivity, and increased mitogenesis, as measured by incorporation of [3H]thymidine. The Fos-like immunoreactivity appeared within 30 min, lasted 2 h and was blocked by the partially selective dopamine D3 receptor compound (+)-UH 232 (cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin). The mitogenic effect, which occurred after a lag time (over 2 h stimulation), was produced with subnanomolar potency and full intrinsic activity by several compounds previously identified as dopamine D2 receptor agonists, e.g. quinpirole, (+)-7-OH-DPAT ((+)-7-hydroxy-2-(di-n-propylamino)tetralin) and RU 24926 (N-n-propyl-di-beta(3-hydroxyphenyl)-ethylamine), and was reversibly blocked by (+)-UH 232 (Ki = 9 nM). Talipexole (B-HT 920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin) was identified as a partial agonist at the dopamine D3 receptor. Dopamine D3 receptor-mediated mitogenesis was potentiated by a phorbol ester and was abolished by pretreatment with pertussis toxin. A mitogenic effect of same amplitude was elicited by bradykinin or carbachol, both acting through constitutive receptors. Bradykinin markedly activated inositol phosphate turnover, and had no effect on forskolin-stimulated cyclic AMP accumulation. Carbachol inhibited forskolin-stimulated cyclic AMP accumulation and had no effect on inositol-phosphate turnover. Quinpirole had no effect on any of these second messenger pathways. Thus, in transfected NG 108-15 cells, the dopamine D3 receptor is coupled to a pertussis toxin-sensitive G protein and mediates two possibly unrelated biological effects, through initial biochemical events that remain to be identified.

Antidromic stimulation of small caliber trigeminal axons causes neurogenic inflammation in the dura mater and tongue as evidenced by marked increases in mast cell activation, protein extravasation, as well as in the numbers of endothelial cytoplasmic vesicles, endothelial microvilli and platelet aggregates within ipsilateral post-capillary venules. In this report, we examined the effects of pretreatment with serotonin1 receptor agonists, dihydroergotamine (50 micrograms/kg, i.v.) and sumatriptan (100 micrograms/kg, i.v.) on the light and electron microscopic changes which develop after trigeminal ganglion stimulation. Both dihydroergotamine and sumatriptan are useful in the acute treatment of vascular headaches and bind with high affinity to 5-HT1D receptors. Both drugs decreased significantly the number of dural vessels showing endothelial or platelet changes and the numbers of activated mast cells, but did not affect the neurogenic response in the tongue. The drugs also blocked the accumulation of horseradish peroxidase reaction product within the endothelium and perivascular space on the stimulated side. The receptor is not present on trigeminovascular fibers innervating extracranial cephalic tissues. Drug mechanism probably involves inhibition of a proximal step in the pathophysiological cascade (e.g., via activation of a prejunctional receptor) because (a) receptors for sumatriptan have not been identified on mast cells whereas the inflammatory response was attenuated in mast cells as well as within platelets and the endothelium and (b) previous work indicates that sumatriptan and dihydroergotamine block neurotransmitter release. Hence, constriction of vascular smooth muscle mediated by postjunctional 5-hydroxytryptamine receptors is unlikely to explain the anti-inflammatory actions of dihydroergotamine or sumatriptan reported here.

Mast cells are involved in allergic reactions, but may also participate in neurogenic inflammation. The morphology of mast cells in rat dura mater and tongue was evaluated by histochemistry, as well as by scanning and transmission electron microscopy following unilateral trigeminal ganglion stimulation (5 min, 5 Hz, 5 ms, and 0.02, 0.1 or 1.0 mA). Mast cells in dura and tongue of normal animals were numerous, perivascular and often in close proximity to nerve fibers. After 5 min of electrical stimulation, mast cells contralateral to the stimulation showed histochemical characteristics of normal peripheral tissue mast cells (Safranin-positive), and by electron microscopy appeared homogeneous with numerous intact electron-dense granules. On the stimulated side, however, the staining characteristics of mast cells showed changes indicating progressive intracellular loss of their granular content. In addition, the total number of stainable mast cells decreased at all three stimulus intensities, but reached significance only at 0.1 and 0.02 mA. Ultrastructural evidence of granule changes consistent with secretion were observed although degranulation was not observed until 20 min after stimulation. There were no mast cell changes after electrical trigeminal stimulation in adult rats treated as neonates with capsaicin to destroy small caliber sensory afferent axons. These results suggest that mast cells may secrete in response to electrical stimulation of trigeminal axons, possibly mediated by antidromic release of neuropeptides, and may participate in the development of neurogenic inflammation.

Mast cells are known to derive from the bone marrow and enter the tissues as immature cells which differentiate under local microenvironmental factors. However, it has not been known how and when these cells enter the brain; moreover, the localization of mast cells in the developing rat brain differs from that of the adult animal. Our anatomical and morphological observations showed that during late embryonic stages and the first 11 days after birth, rat brain mast cells were exclusively concentrated within the pia mater surrounding the diencephalon, the choroid fissure and within the choroid plexus. Histochemically these cells contained only a few toluidine blue metachromatic granules, suggesting a 'mucosal' phenotype and the absence of heparin. Later, during a transitional phase from postnatal day 11 to 13, these cells migrated along blood vessels of the fimbria, the hippocampus and the penetrating vessels of the thalamus into the dorsolateral and posterolateral thalamic nuclei. These cells contained more metachromatic granules, and from day 13 on, they assumed their adult perivascular localization within the thalamus with numerous metachromatic granules similar to those described for mature thalamic and serosal mast cells.

Mast cells are involved in allergic reactions where they secrete numerous mediators in response to immunoglobulin E and antigen. However, they have recently been implicated in neuroinflammatory conditions with a higher prevalence in women, and there have been clinical reports of progesterone anaphylaxis. When tested on purified rat peritoneal mast cells, progesterone alone stimulated release only of 5-hydroxytryptamine (serotonin) in a dose- and time-dependent manner. Serotonin release by progesterone was exceptional because it was not accompanied by histamine release or degranulation and was either augmented or unaffected by drugs which inhibit secretion induced by the classic mast cell secretagogue, compound 48/80. These findings indicate that mast cells are capable of selective serotonin secretion, previously shown only after pretreatment with certain tricyclic drugs, and may be involved in neuroendocrine syndromes.

This study was performed to determine to what extent the morphology of the rabbit middle cerebral artery is affected by the absence of the sympathetic nervous system. Six weeks after unilateral ablation of the superior cervical ganglion, which induced ipsilateral degeneration and disappearance of the perivascular noradrenergic nerve fibers, comparison between the ipsi- and the contralateral middle cerebral arteries revealed that the denervated arterial wall underwent significant thickening. This thickening was principally due to hypertrophy of the smooth muscle cells (SMC), together with an increase in the amount of medial and adventitial collagen. The hypertrophied SMC showed important morphological and ultrastructural modifications--irregular shape, increase in the number of organelles (particularly of Golgi apparatus, free ribosomes, rough endoplasmic reticulum and microtubules), large indented nuclei rich in euchromatin--indicating profound changes in their metabolic and contractile activity which could result in an alteration of their mechanical properties. As these alterations were strictly ipsilateral to the sympathectomy it is likely that they are the direct consequence of the suppression of a regulatory 'trophic' factor linked to the presence of sympathetic nerve fibers. This concept is reinforced by the fact that the first SMC affected are those situated at the media/adventitial border, in the vicinity of adventitial nerve bundles. Thus, the sympathetic nervous system appears to play a key role in the long-term regulation of the cerebral vascular tree structure.