CRF receptors in the rodent and human cardiovascular systems: Species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine or [(125)I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF(2) receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF(2) receptors were detected with (125)I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with (125)I-[Tyr(0), Glu(1), Nle(17)]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF(2) receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.

Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists

The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst2), somatostatin receptor subtype 3 (sst3), and somatostatin receptor subtype 5 (sst5) in vitro at the protein level. METHODS: Human embryonic kidney 293 (HEK293) cells expressing the sst2, sst3, or the sst5 were used in a morphologic immunocytochemical internalization assay using specific sst2, sst3 and sst5 antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst2-expressing CHO-K1 cells using an ELISA. RESULTS: Agonists with a high sst2-binding affinity were able to induce sst2 internalization in the HEK293 and CHO-K1 cell lines. New sst2 agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr3, Thr8]-octreotide; NOC is [1-NaI3]-octreotide; and BOC-ATE is [BzThi3, Thr8]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst2 than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst3 affinity such as KE108 were able to induce sst3 internalization. In sst2- or sst3-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst2- or sst3-selective antagonists, respectively. Antagonists alone had no effect on sst2 or sst3 internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst5 internalization. Unexpectedly, however, potent sst5 agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst5 internalization under the same conditions. CONCLUSION: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst2, sst3, and sst5 internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst2 and sst3 internalization, sst5 internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst5 agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

Novel 111In-labelled bombesin analogues for molecular imaging of prostate tumours

PURPOSE: It has been shown that some primary human tumours and their metastases, including prostate and breast tumours, overexpress gastrin-releasing peptide (GRP) receptors. Bombesin (BN) is a neuropeptide with a high affinity for these GRP receptors. We demonstrated successful scintigraphic visualisation of BN receptor-positive tumours in preclinical studies using the radiolabelled BN analogue [(111)In-DTPA-Pro(1),Tyr(4)]BN. However, the receptor affinity as well as the serum stability of this analogue leave room for improvement. Therefore new (111)In-labelled BN analogues were synthesised and evaluated in vitro and in vivo. METHODS AND RESULTS: The receptor affinity of the new BN analogues was tested on human GRP receptor-expressing prostate tumour xenografts and rat colon sections. Analogues with high receptor affinity (low nM range) were selected for further evaluation. Incubation in vitro of GRP receptor-expressing rat CA20948 and human PC3 tumour cells with the (111)In-labelled analogues resulted in rapid receptor-mediated uptake and internalisation. The BN analogue with the best receptor affinity and in vitro internalisation characteristics, Cmp 3 ([(111)In-DTPA-ACMpip(5),Tha(6),betaAla(11),Tha(13),Nle(14)]BN(5-14)), was tested in vivo in biodistribution studies using rats bearing GRP receptor-expressing CA20948 tumours, and nude mice bearing human PC3 xenografts. Injection of (111)In-labelled Cmp 3 in these animals showed high, receptor-mediated uptake in receptor-positive organs and tumours which could be visualised using planar gamma camera and microSPECT/CT imaging. CONCLUSION: With their enhanced receptor affinity and their rapid receptor-mediated internalisation in vitro and in vivo, the new BN analogues, and especially Cmp 3, are promising candidates for use in diagnostic molecular imaging and targeted radionuclide therapy of GRP receptor-expressing cancers.

Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals?

PURPOSE: Gallium-68 is a metallic positron emitter with a half-life of 68 min that is ideal for the in vivo use of small molecules, such as [68Ga-DOTA,Tyr3]octreotide, in the diagnostic imaging of somatostatin receptor-positive tumours. In preclinical studies it has shown a striking superiority over its 111In-labelled congener. The purpose of this study was to evaluate whether third-generation somatostatin-based, radiogallium-labelled peptides show the same superiority. METHODS: Peptides were synthesised on solid phase. The receptor affinity was determined by in vitro receptor autoradiography. The internalisation rate was studied in AR4-2J and hsst-HEK-transfected cell lines. The pharmacokinetics was studied in a rat xenograft tumour model, AR4-2J. RESULTS: All peptides showed high affinities on hsst2, with the highest affinity for the Ga(III)-complexed peptides. On hsst3 the situation was reversed, with a trend towards lower affinity of the Ga(III) peptides. A significantly increased internalisation rate was found in sst2-expressing cells for all 67Ga-labelled peptides. Internalisation into HEK-sst3 was usually faster for the 111In-labelled peptides. No internalisation was found into sst5. Biodistribution studies employing [67Ga-DOTA,1-Nal3]octreotide in comparison to [111In-DOTA,1-Nal3]octreotide and [67Ga-DOTA,Tyr3]octreotide showed a significantly higher and receptor-mediated uptake of the two 67Ga-labelled peptides in the tumour and somatostatin receptor-positive tissues. A patient study illustrated the potential advantage of a broad receptor subtype profile radiopeptide over a high-affinity sst2-selective radiopeptide. CONCLUSION: This study demonstrates that 67/68Ga-DOTA-octapeptides show distinctly better preclinical, pharmacological performances than the 111In-labelled peptides, especially on sst2-expressing cells and the corresponding animal models. They may be excellent candidates for further development for clinical studies.

Intraindividual comparison of human radial and internal thoracic arteries in vitro and the effect of preoperative calcium blocker therapy

The patency rate of radial artery (RA) conduits is considerably lower than that of internal thoracic artery (ITA) grafts and the evidence suggests that this is due to a clinically suspected higher incidence of vasospasm. The aim of this study was, therefore, to compare intraindividually the pharmacological reactivity of RA with that of ITA. Both RA and ITA were taken from the same patients and investigated in parallel. Changes in tone were monitored isometrically on ring preparations from both arteries in organ baths with modified Krebs-Henseleit solution containing 1.25 mm calcium chloride at 1 g passive preload. In intraindividual comparisons maximal receptor-mediated contractile responses to noradrenaline and endothelin-1 and endothelium-dependent acetylcholine-induced relaxant responses revealed no differences between both arteries. By contrast, depolarization-induced contractions to potassium chloride (KCl) appeared to be significantly higher in RA than in ITA. Further analysis, however, revealed that this difference was due to preoperative calcium entry blocker (Ca(2+)eB) therapy. Compared with control tissues, maximal responses to KCl were significantly attenuated in the ITA but unchanged in RA when arteries were obtained from patients with preoperative Ca(2+)eB therapy. The present results suggested that functional responses to pharmacological stimuli of both RA and ITA were quite similar. Preoperative Ca(2+)eB therapy, however, attenuated markedly responses to KCl of the ITA leaving those of RA unchanged. These results, demonstrating a lower sensitivity to Ca(2+)eB of RA, therefore suggested that in addition to Ca(2+)eB other classes of drug may be more effective at reducing the propensity of RA conduits to vasospasm.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Immunomodulatory lipids in plants: plant fatty acid amides and the human endocannabinoid system

Since the discovery that endogenous lipid mediators show similar cannabimimetic effects as phytocannabinoids from CANNABIS SATIVA, our knowledge about the endocannabinoid system has rapidly expanded. Today, endocannabinoid action is known to be involved in various diseases, including inflammation and pain. As a consequence, the G-protein coupled cannabinoid receptors, endocannabinoid transport, as well as endocannabinoid metabolizing enzymes represent targets to block or enhance cannabinoid receptor-mediated signalling for therapeutic intervention. Based on the finding that certain endocannabinoid-like fatty acid N-alkylamides from purple coneflower ( ECHINACEA spp.) potently activate CB2 cannabinoid receptors we have focused our interest on plant fatty acid amides (FAAs) and their overall cannabinomodulatory effects. Certain FAAs are also able to partially inhibit the action of fatty acid amide hydrolase (FAAH), which controls the breakdown of endocannabinoids. Intriguingly, plants lack CB receptors and do not synthesize endocannabinoids, but express FAAH homologues capable of metabolizing plant endogenous N-acylethanolamines (NAEs). While the site of action of these NAEs in plants is unknown, endogenous NAEs and arachidonic acid glycerols in animals interact with distinct physiological lipid receptors, including cannabinoid receptors. There is increasing evidence that also plant FAAs other than NAEs can pharmacologically modulate the action of these endogenous lipid signals. The interference of plant FAAs with the animal endocannabinoid system could thus be a fortunate evolutionary cross point with yet unexplored therapeutic potential.

Virosomes can enter cells by non-phagocytic mechanisms

Phagocytosis of fine particles (1 mum) by macrophages is a ligand-receptor-mediated, actin-based process, whereas the entering of smaller particles (

Human internal thoracic arteries from diabetic patients are resistant to endothelial dysfunction

The aim of this analysis was to compare vasoreactive properties of internal thoracic arteries (ITA) grafts from diabetic (DM) to those of non-diabetic (ND) patients. Ring segments of ITA, taken from patients undergoing coronary artery bypass grafting, were suspended in organ bath chambers filled with modified Krebs-Henseleit solution and contractile responses to potassium chloride (KCl), noradrenaline (NA), endothelin-1 (ET-l), and endothelium-dependent relaxant responses to acetylcholine (ACH) were recorded isometrically. The receptor-mediated agonists NA and ET-1 stimulated ITA from both groups within similar concentration ranges while ITA from DM patients proved to be significantly more sensitive to KCl than ITA from ND. Furthermore, maximal contractile responses indicated that KCl (3.79 +/- 0.30 g, n = 7 in DM and 2.50 +/- 0.23 g, n = 29 in ND, P < 0.05) evoked significantly higher responses in ITA from DM as compared to the ND control group while both NA and ET-l stimulated ITA from both groups with similar efficacies. Endothelium-dependent relaxant responses to ACH proved to be similar in both groups when expressed as percentages of the pre-existing tone. The present data support the contention that in comparison to ND controls arteries from DM patients are more sensitive to depolarization but endothelial dysfunction is less frequent in human ITA than expected from observations in systemic vascular beds.

Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Quantification of gold nanoparticle cell uptake under controlled biological conditions and adequate resolution

Aim: We examined cellular uptake mechanisms of fluorescently labeled polymer-coated gold nanoparticles (NPs) under different biological conditions by two quantitative, microscopic approaches. Materials & methods: Uptake mechanisms were evaluated using endocytotic inhibitors that were tested for specificity and cytotoxicity. Cellular uptake of gold NPs was analyzed either by laser scanning microscopy or transmission electron microscopy, and quantified by means of stereology using cells from the same experiment. Results: Optimal inhibitor conditions were only achieved with chlorpromazine (clathrin-mediated endocytosis) and methyl-β-cyclodextrin (caveolin-mediated endocytosis). A significant methyl-β-cyclodextrin-mediated inhibition (63-69%) and chlorpromazine-mediated increase (43-98%) of intracellular NPs was demonstrated with both imaging techniques, suggesting a predominant uptake via caveolin-medicated endocytois. Transmission electron microscopy imaging revealed more than 95% of NPs localized in intracellular vesicles and approximately 150-times more NP events/cell were detected than by laser scanning microscopy. Conclusion: We emphasize the importance of studying NP-cell interactions under controlled experimental conditions and at adequate microscopic resolution in combination with stereology. Original submitted 10 July 2012; Revised submitted 23 January 2013.

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis.

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.

Structure-function relation in retinoid regulated gene expression

Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^