Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.

PTSD, Depression, and Substance Abuse Symptoms in Women Exposed to Intimate Partner Abuse

The present study explores relationships among several established correlates of trauma in women exposed to intimate partner abuse (IPA), including PTSD, depression, and dissociation symptoms as well as alcohol use as well as other trauma-related variables, such as social support and violence exposure. Two analysis methods were utilized: variable-oriented methods, which examine relationships between variables, and person-oriented analysis methods, which examine groupings of participants within a larger sample (N = 233). Results of the variable-oriented analyses indicated positive links among depression, PTSD, dissociation, and alcohol use in women exposed to IPA, as well as positive links between the aforementioned psychological symptoms and exposure to violence. Social support was related to decreased psychological symptoms. Person-oriented analyses indicated the presence of four unique profiles of women within the larger study sample: Profile 1 (n = 21), which was labeled High Dissociation, Low Depression/PTSD; Profile 2 (n = 150), which was labeled Low Symptoms, High Social Support, Profile 3 (n = 41); which was labeled Low Dissociation, High Depression/PTSD; and Profile 4 (n = 22), which was labeled High Symptoms, Low Social Support. This research supports previous findings about the relationships among several variables related to IPA as well as suggests the need for careful consideration of differences among women within the larger context of research, advocacy, and clinical interventions related to IPA.

Treatment Outcomes for Eating Disorder Patients With and Without Comorbid Substance Abuse Diagnoses

The impact of comorbid substance abuse and eating disorder diagnoses in an eating disorder treatment facility remains uncertain. Recent data suggest that in a substance abuse treatment setting, patients with comorbid eating disorders fared less favorably than patients without a comorbid diagnosis (Cohen et al., 2010; Glasner-Edwards et al., 2011). The purpose of this study is to compare eating disorder symptoms over the course of treatment for patients with and without comorbid substance abuse diagnoses in an eating disorder treatment facility. Archival data from an eating disorder treatment facility was used. Twenty-seven women with comorbid eating disorder and substance abuse diagnoses (EDSUD) were compared to twenty-seven women with an eating disorder diagnosis (ED) only. The subjects were compared on three scales from the Eating Disorder Inventory-III (EDI-3) by group, and pre- and post-treatment. The scales were Personal Alienation (PA), Interoceptive Deficits (ID), and Emotional Dysregulation (EmD). There was a significant decrease in symptoms post-treatment for all subjects on the PA and ID scales, and there was a significant difference between the EDSUD subjects and ED subjects on two scales. EDSUD subjects fared significantly less favorably on the ID and EmD scales. Women with EDSUD report more symptoms of Interoceptive Deficits and Emotional Dysregulation when compared to women with an ED diagnosis and no comorbid substance use. Subjects benefited from treatment in terms of less Personal Alienation and Interoceptive Deficits.

Acceptance and Commitment Therapy: Increasing Psychologists' Willingness to Assess and Treat Substance-abusing Clients.

The high prevalence of substance abuse in the United States and the low rates of assessment and treatment of these disorders by mental health providers points to a growing need to understand the factors that prevent substance-abusing individuals from receiving adequate services. Psychologists are one group of mental health providers that show little interest in working with this population and receive little research attention on the topic. This paper explores the potential role that education, previous experience, and the impact that holding stigmatizing beliefs towards substance-abusing individuals has on psychologists' willingness to provide clinical services for clients struggling with addiction. Acceptance and Commitment Therapy (ACT) is explored as a potential intervention for psychologists.

From Structure to Substance: Has the Constitutional Treaty improved the Chances for a Common Foreign and Security Policy? Research Papers in Law, 6/2005

From the Introduction. A common foreign and security policy for the European Union is an issue of the day. While most academic and many political observers believe that it would be in the interest of the Union to have a common policy, there is quite some disagreement as to how this is to be achieved and whether it should be accomplished in an assured and regular manner or whether it should come about on an ad hoc basis only when it is in the clear interest of all member states at any particular time. In other words, is a common foreign policy to be a fundamental characteristic of the Union or is it to be an occasional occurrence when advantageous and convenient, the ‘C’ in CFSP – as one observer has sarcastically commented – standing not for ‘Common’ but for ‘Convenient’?2

The Impact of a Substance Abuse Treatment for Offenders in Relation to the Time Spent in Treatment

The objective of this research is to evaluate the outcomes of a treatment for addicts. 123 subjects were tested before treatment and at 5, 8 and 11 months follow-up periods with a French version of the Addiction Severity Index (ASI). Exposure to treatment was based on the number of clients’ contact-hours with a therapist. The sample was divided into three groups according to the number of hours spent in treatment. The data was analysed using MANOVA on the seven scales of the ASI for the three groups and the four time periods. Results showed that all groups improved significantly but that this improvement was not related to the number of hours spent in treatment.