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Together, Nobel Prize honoured work, mathematics, physics and the laws of nature have drawn a concept of clockwise cycling carboxylic acids in Krebs' Citric Acid Cycle. A Citric Acid Cycle complex is defined by specific substrate, product and regulation. Recently, the Citric Acid Cycle 1.1 complex was introduced as an NAD + -regulated cycle with the substrate, lactic acid and the product, malic acid. Here, we introduce the concept of the Citric Acid Cycle 2.1 complex as an FAD-regulated cycle with the substrate, malic acid and the products, succinic acid or citric acid. The function of the Citric Acid Cycle 2.1 complex is to balance stress situations within the cell. We propose that the biological function of Citric Acid Cycle 2.1 in muscles is to accelerate recovery of ATP; whereas in white tissue adipocytes our testing of the theoretical concept led to the storage of energy as lipids., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

Schizophrenia is a chronic and severe mental disorder that affects over 20 million people worldwide. Common symptoms include distortions in thinking, perception, emotions, language, and self awareness. Different hypotheses have been proposed to explain the development of schizophrenia, however, there are no unifying features between the proposed hypotheses. Schizophrenic patients have perturbed levels of glucose in their cerebrospinal fluid, indicating a disturbance in glucose metabolism. We have explored the possibility that disturbances in glucose metabolism can be a general mechanism for predisposition and manifestation of the disease. We discuss glucose metabolism as a network of signaling pathways. Glucose and glucose metabolites can have diverse actions as signaling molecules, such as regulation of transcription factors, hormone and cytokine secretion and activation of neuronal cells, such as microglia. The presented model challenges well-established concepts in enzyme kinetics and glucose metabolism. We have developed a 'two-cell' model of glucose metabolism, which can explain the effects of electroconvulsive therapy and the beneficial and side effects of olanzapine treatment. Arrangement of glycolytic enzymes into metabolic signaling complexes within the 'two hit' hypothesis, allows schizophrenia to be formulated in two steps. The 'first hit' is the dysregulation of the glucose signaling pathway. This dysregulation of glucose metabolism primes the central nervous system for a pathological response to a 'second hit' via the astrocytic glycogenolysis signaling pathway., (© 2021. The Author(s).)

In 1937, Sir H. A Krebs first published the Citric Acid Cycle, a unidirectional cycle with carboxylic acids. The original concept of the Citric Acid Cycle from Krebs' 1953 Nobel Prize lecture illustrates the unidirectional degradation of lactic acid to water, carbon dioxide and hydrogen. Here, we add the heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex, connecting the original Citric Acid Cycle to the flow of energy and material. The heart lactate dehydrogenase•proton-linked monocarboxylate transporter 1 complex catalyses the first reaction of the Citric Acid Cycle, the oxidation of lactate to pyruvate, and thus secures the provision of pyruvic acid. In addition, we modify Krebs' original concept by feeding the cycle with oxaloacetic acid. Our concept enables the integration of anabolic processes and allows adaption of the organism to recover ATP faster.

Medical biobanking is concerned with establishing and maintaining large-scale repositories of biological specimens combined with comprehensive archives of clinical and biographical information on donors. This aims for controlled high and consistent quality of specimens for future biomedical research. One major objective is to assemble multiple blood components for various types of biochemical analysis and experimentation including different isolated cell types. With proper cryo-conservation, blood-derived cells can be conserved and revitalized after thawing and employed as in-vitro cell models carrying specific biological traits of donors. Optimizing pre-analytical methods can reduce pre-analytical variance thereby reducing imprecision of analytical data. This is particularly valuable for multivariate analyses of biological systems ("omics") and biomarker research. Introducing biobanking to psychiatry carries the challenge of making diagnostic allocation more compatible with biological entities than is achieved with current diagnostic categories of ICD-10 or DSM-V. Diagnostic or transdiagnostic subgroups can be stratified using biologically anchored clinical criteria. An important ethical issue of biobanking is the need for broad consent by the donors for specimen use in not yet defined future research projects. The organizational, logistic and financial costs of establishing and maintaining a biobank are considerable, but seem well warranted in view of the gainable advances in biomedical research quality, translations and clinical applications., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme. All rights reserved.)

Since formulation of the Astrocyte-Neuron Lactate Shuttle (ANLS) hypothesis in 1994, the hypothesis has provoked criticism and debate. Our review does not criticise, but rather integrates experimental data characterizing proton-linked monocarboxylate transporters (MCTs) into the ANLS. MCTs have wide substrate specificity and are discussed to be in protein complex with a proton donor (PD). We particularly focus on the proton-driven transfer of l-lactic acid (l-lacH) and pyruvic acid (pyrH), were PDs link MCTs to a flow of energy. The precise nature of the PD predicts the activity and catalytic direction of MCTs. By doing so, we postulate that the MCT4·phosphoglycerate kinase complex exports and at the same time in the same astrocyte, MCT1·carbonic anhydrase II complex imports monocarboxylic acids. Similarly, neuronal MCT2 preferentially imports pyrH. The repertoire of MCTs in astrocytes and neurons allows them to communicate via monocarboxylic acids. A change in imported pyrH/l-lacH ratio in favour of l-lacH encodes signals stabilizing the transit of glucose from astrocytes to neurons. The presented astrocyte neuron communication hypothesis has the potential to unite the community by suggesting that the exchange of monocarboxylic acids paves the path of glucose provision., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (© 2020 the Author(s), licensee AIMS Press.)

The Embden-Meyerhof-Parnas (EMP) pathway comprises eleven cytosolic enzymes interacting to metabolize glucose to lactic acid [CH 3 CH(OH)COO H ]. Glycolysis is largely considered as the conversion of glucose to pyruvate (CH 3 COCOO - ). We consider glycolysis to be a cellular process and as such, transporters mediating glucose uptake and lactic acid release and enable the flow of metabolites through the cell, must be considered as part of the EMP pathway. In this review, we consider the flow of metabolites to be coupled to a flow of energy that is irreversible and sufficient to form ordered structures. This latter principle is highlighted by discussing that lactate dehydrogenase (LDH) complexes irreversibly reduce pyruvate/H + to lactate [CH 3 CH(OH)COO - ], or irreversibly catalyze the opposite reaction, oxidation of lactate to pyruvate/H + . However, both LDH complexes are considered to be driven by postulated proton transport chains. Metabolism of glucose to two lactic acids is introduced as a unidirectional, continuously flowing pathway. In an organism, cell membrane-located proton-linked monocarboxylate transporters catalyze the final step of glycolysis, the release of lactic acid. Consequently, both pyruvate and lactate are discussed as intermediate products of glycolysis and substrates of regulated crosscuts of the glycolytic flow.

De-regulation of energy metabolism by the dopaminergic system is linked to neurological diseases such as schizophrenia and bipolar disorder. Inverse agonists are thought to be more beneficial in treating neurological diseases than neutral antagonists, but only limited experimental data are available regarding the impact of constitutive signalling on energy metabolism. The aim of the present study was to assess the impact of constitutive dopamine-1 receptor (D1R) and dopamine-5 receptor (D5R) signalling on downstream targets in transiently and stably transfected HEK293T cells. The high constitutive activity of D5R was accompanied by increased Na(+)/H(+) exchanger (NHE) activity and accelerated glucose degradation due to increased transcription and translation of the Na, K-ATPase-α3 and NHE-2. Chronic treatment with an agonist increased the mRNA levels of the α2 Na,K-ATPase, NHE-2 and NHE-3. Constitutive D5R activation of a cAMP response element-based reporter was regulated by G protein-coupled receptor kinase 2, but this did not affect the cell-surface abundance of the receptor. Our data suggest that constitutive and agonist-induced activity of D5R differentially regulates the activity and expression of proteins., (© 2014 FEBS.)

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Neuropeptide signaling at the cell surface is regulated by metalloendopeptidases, which degrade peptides in the extracellular fluid, and beta-arrestins, which interact with G protein-coupled receptors (GPCRs) to mediate desensitization. beta-Arrestins also recruit GPCRs and mitogen-activated protein kinases to endosomes to allow internalized receptors to continue signaling, but the mechanisms regulating endosomal signaling are unknown. We report that endothelin-converting enzyme-1 (ECE-1) degrades substance P (SP) in early endosomes of epithelial cells and neurons to destabilize the endosomal mitogen-activated protein kinase signalosome and terminate signaling. ECE-1 inhibition caused endosomal retention of the SP neurokinin 1 receptor, beta-arrestins, and Src, resulting in markedly sustained ERK2 activation in the cytosol and nucleus, whereas ECE-1 overexpression attenuated ERK2 activation. ECE-1 inhibition also enhanced SP-induced expression and phosphorylation of the nuclear death receptor Nur77, resulting in cell death. Thus, endosomal ECE-1 attenuates ERK2-mediated SP signaling in the nucleus to prevent cell death. We propose that agonist availability in endosomes, here regulated by ECE-1, controls beta-arrestin-dependent signaling of endocytosed GPCRs.

Somatostatin receptor (SSTR) endocytosis influences cellular responsiveness to agonist stimulation and somatostatin receptor scintigraphy, a common diagnostic imaging technique. Recently, we have shown that SSTR1 is differentially regulated in the endocytic and recycling pathway of pancreatic cells after agonist stimulation. Additionally, SSTR1 accumulates and releases internalized somatostatin-14 (SST-14) as an intact and biologically active ligand. We also demonstrated that SSTR2A was sequestered into early endosomes, whereas internalized SST-14 was degraded by endosomal peptidases and not routed into lysosomal degradation. Here, we examined the fate of peptide agonists in rat insulinoma cells expressing SSTR3 by biochemical methods and confocal laser scanning microscopy. We found that [(125)I]Tyr11-SST-14 rapidly accumulated in intracellular vesicles, where it was degraded in an ammonium chloride-sensitive manner. In contrast, [(125)I]Tyr1-octreotide accumulated and was released as an intact peptide. Rhodamine-B-labeled SST-14, however, was rapidly internalized into endosome-like vesicles, and fluorescence signals colocalized with the lysosomal marker protein cathepsinD. Our data show that SST-14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an intact peptide. Chronic stimulation of SSTR3 also induced time-dependent downregulation of the receptor. Thus, the intracellular processing of internalized SST-14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes.

Agonist-induced internalization of somatostatin receptors (ssts) determines subsequent cellular responsiveness to peptide agonists and influences sst receptor scintigraphy. To investigate sst2A trafficking, rat sst2A tagged with epitope was expressed in human embryonic kidney cells and tracked by antibody labeling. Confocal microscopical analysis revealed that stimulation with sst and octreotide induced internalization of sst2A. Internalized sst2A remained sequestrated within early endosomes, and 60 min after stimulation, internalized sst2A still colocalized with beta-arrestin1-enhanced green fluorescence protein (EGFP), endothelin-converting enzyme-1 (ECE-1), and rab5a. Internalized (125)I-Tyr(11)-SST-14 was rapidly hydrolyzed by endosomal endopeptidases, with radioactive metabolites being released from the cell. Internalized (125)I-Tyr(1)-octreotide accumulated as an intact peptide and was released from the cell as an intact peptide ligand. We have identified ECE-1 as one of the endopeptidases responsible for inactivation of internalized SST-14. ECE-1-mediated cleavage of SST-14 was inhibited by the specific ECE-1 inhibitor, SM-19712, and by preventing acidification of endosomes using bafilomycin A(1). ECE-1 cleaved SST-14 but not octreotide in an acidic environment. The metallopeptidases angiotensin-1 converting enzyme and ECE-2 did not hydrolyze SST-14 or octreotide. Our results show for the first time that stimulation with SST-14 and octreotide induced sequestration of sst2A into early endosomes and that endocytosed SST-14 is degraded by endopeptidases located in early endosomes. Furthermore, octreotide was not degraded by endosomal peptidases and was released as an intact peptide. This mechanism may explain functional differences between octreotide and SST-14 after sst2A stimulation. Moreover, further investigation of endopeptidase-regulated trafficking of neuropeptides may result in novel concepts of neuropeptide receptor inactivation in cancer diagnosis.

Proteinase-activated receptor-2 (PAR2) is a seven transmembrane spanning, G-protein-coupled receptor, present on the membrane of many cell types including keratinocytes. In skin, PAR2 is suggested to play a regulatory role during inflammation, epidermal barrier function, and pruritus. PAR2 is activated by trypsin-like proteases by a unique mechanism where cleavage of the receptor leads to the release of a small peptide, which activates the receptor as a tethered ligand. The endogenous activators of PAR2 on keratinocytes have not been identified as of yet. Potential candidates are kallikrein-related peptidases (KLKs) expressed by epidermal cells. Therefore, the ability of four human skin-derived KLKs was examined with regard to their capacity to activate PAR2 in vitro. PAR2 cleavage was followed by immunofluorescence analysis and functional activation by measurements of changes in intracellular calcium levels. We found that KLK5 and KLK14, but neither KLK7 nor KLK8, induced PAR2 signalling. We conclude that certain, but not all, epidermal KLKs are capable of activating PAR2. We could also show the coexpression of KLK14 and PAR2 receptor in inflammatory skin disorders. These in vitro results suggest that KLKs may take part in PAR2 activation in the epidermis and thereby in PAR2-mediated inflammatory responses, including epidermal barrier repair and pruritus. The role of KLKs in PAR2 activation in vivo remains to be elucidated.

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

Neuropeptide signaling requires the presence of G protein-coupled receptors (GPCRs) at the cell surface. Activated GPCRs interact with beta-arrestins, which mediate receptor desensitization, endocytosis, and mitogenic signaling, and the peptide-receptor-arrestin complex is sequestered into endosomes. Although dissociation of beta-arrestins is required for receptor recycling and resensitization, the critical event that initiates this process is unknown. Here we report that the agonist availability in the endosomes, controlled by the membrane metalloendopeptidase endothelin-converting enzyme 1 (ECE-1), determines stability of the peptide-receptor-arrestin complex and regulates receptor recycling and resensitization. Substance P (SP) binding to the tachykinin neurokinin 1 receptor (NK1R) induced membrane translocation of beta-arrestins followed by trafficking of the SP-NK1R-beta-arrestin complex to early endosomes containing ECE-1a-d. ECE-1 degraded SP in acidified endosomes, disrupting the complex; beta-arrestins returned to the cytosol, and the NK1R, freed from beta-arrestins, recycled and resensitized. An ECE-1 inhibitor, by preventing NK1R recycling in endothelial cells, inhibited resensitization of SP-induced inflammation. This mechanism is a general one because ECE-1 similarly regulated NK3R resensitization. Thus, peptide availability in endosomes, here regulated by ECE-1, determines the stability of the peptide-receptor-arrestin complex. This mechanism regulates receptor recycling, which is necessary for sustained signaling, and it may also control beta-arrestin-dependent mitogenic signaling of endocytosed receptors. We propose that other endosomal enzymes and transporters may similarly control the availability of transmitters in endosomes to regulate trafficking and signaling of GPCRs. Antagonism of these endosomal processes represents a strategy for inhibiting sustained signaling of receptors, and defects may explain the tachyphylaxis of drugs that are receptor agonists.

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.

This review focuses on the role of the peripheral nervous system in cutaneous biology and disease. During the last few years, a modern concept of an interactive network between cutaneous nerves, the neuroendocrine axis, and the immune system has been established. We learned that neurocutaneous interactions influence a variety of physiological and pathophysiological functions, including cell growth, immunity, inflammation, pruritus, and wound healing. This interaction is mediated by primary afferent as well as autonomic nerves, which release neuromediators and activate specific receptors on many target cells in the skin. A dense network of sensory nerves releases neuropeptides, thereby modulating inflammation, cell growth, and the immune responses in the skin. Neurotrophic factors, in addition to regulating nerve growth, participate in many properties of skin function. The skin expresses a variety of neurohormone receptors coupled to heterotrimeric G proteins that are tightly involved in skin homeostasis and inflammation. This neurohormone-receptor interaction is modulated by endopeptidases, which are able to terminate neuropeptide-induced inflammatory or immune responses. Neuronal proteinase-activated receptors or transient receptor potential ion channels are recently described receptors that may have been important in regulating neurogenic inflammation, pain, and pruritus. Together, a close multidirectional interaction between neuromediators, high-affinity receptors, and regulatory proteases is critically involved to maintain tissue integrity and regulate inflammatory responses in the skin. A deeper understanding of cutaneous neuroimmunoendocrinology may help to develop new strategies for the treatment of several skin diseases.

Transient stimulation with substance P (SP) induces endocytosis and recycling of the neurokinin-1 receptor (NK(1)R). The effects of sustained stimulation by high concentrations of SP on NK(1)R trafficking and Ca(2+) signaling, as may occur during chronic inflammation and pain, are unknown. Chronic exposure to SP (100 nm, 3 h) completely desensitized Ca(2+) signaling by wild-type NK(1)R (NK(1)Rwt). Resensitization occurred after 16 h, and cycloheximide prevented resensitization, implicating new receptor synthesis. Lysine ubiquitination of G-protein-coupled receptors is a signal for their trafficking and degradation. Lysine-deficient mutant receptors (NK(1)RDelta5K/R, C-terminal tail lysines; and NK(1)RDelta10K/R, all intracellular lysines) were expressed at the plasma membrane and were functional because they responded to SP by endocytosis and by mobilization of Ca(2+) ions. SP desensitized NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. However, NK(1)RDelta5K/R and NK(1)RDelta10K/R resensitized 4-8-fold faster than NK(1)Rwt by cycloheximide-independent mechanisms. NK(1)RDelta325 (a naturally occurring truncated variant) showed incomplete desensitization, followed by a marked sensitization of signaling. Upon labeling receptors in living cells using antibodies to extracellular epitopes, we observed that SP induced endocytosis of NK(1)Rwt, NK(1)RDelta5K/R, and NK(1)RDelta10K/R. After 4 h in SP-free medium, NK(1)RDelta5K/R and NK(1)RDelta10K/R recycled to the plasma membrane, whereas NK(1)Rwt remained internalized. SP induced ubiquitination of NK(1)Rwt and NK(1)RDelta5K/R as determined by immunoprecipitation under nondenaturing and denaturing conditions and detected with antibodies for mono- and polyubiquitin. NK(1)RDelta10K/R was not ubiquitinated. Whereas SP induced degradation of NK(1)Rwt, NK(1)RDelta5K/R and NK(1)RDelta10K/R showed approximately 50% diminished degradation. Thus, chronic stimulation with SP induces ubiquitination of the NK(1)R, which mediates its degradation and down-regulation.

Calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene related peptide (CGRP) and intermedin. Although CGRP is widely expressed in the nervous system, less is known about the localization of CLR and RAMP1. To localize these proteins, we raised antibodies to CLR and RAMP1. Antibodies specifically interacted with CLR and RAMP1 in HEK cells coexpressing rat CLR and RAMP1, determined by Western blotting and immunofluorescence. Fluorescent CGRP specifically bound to the surface of these cells and CGRP, CLR, and RAMP1 internalized into the same endosomes. CLR was prominently localized in nerve fibers of the myenteric and submucosal plexuses, muscularis externa and lamina propria of the gastrointestinal tract, and in the dorsal horn of the spinal cord of rats. CLR was detected at low levels in the soma of enteric, dorsal root ganglia (DRG), and spinal neurons. RAMP1 was also localized to enteric and DRG neurons and the dorsal horn. CLR and RAMP1 were detected in perivascular nerves and arterial smooth muscle. Nerve fibers containing CGRP and intermedin were closely associated with CLR fibers in the gastrointestinal tract and dorsal horn, and CGRP and CLR colocalized in DRG neurons. Thus, CLR and RAMP1 may mediate the effects of CGRP and intermedin in the nervous system. However, mRNA encoding RAMP2 and RAMP3 was also detected in the gastrointestinal tract, DRG, and dorsal horn, suggesting that CLR may associate with other RAMPs in these tissues to form a receptor for additional peptides such as adrenomedullin., ((c) 2005 Wiley-Liss, Inc.)

Mesenchymal stem cells (MSCs) are capable of down-regulating alloimmune responses and promoting the engraftment of hematopoietic stem cells. MSCs may therefore be suitable for improving donor-specific tolerance induction in solid-organ transplantation. Cells from cadaveric vertebral bone marrow (V-BM), aspirated iliac crest-BM, and peripheral blood progenitor cells were compared. Cells were characterized by flow cytometry and colony assays. MSCs generated from V-BM were assayed for differentiation capacity and immunomodulatory function. A median 5.7 x 10(8) nucleated cells (NCs) were recovered per vertebral body. The mesenchymal progenitor, colony-forming unit-fibroblast, frequency in V-BM (11.6/10(5) NC, range: 6.0-20.0) was considerably higher than in iliac crest-BM (1.4/10(5) NC, range: 0.4-2.6) and peripheral blood progenitor cells (not detectable). MSC generated from V-BM had the typical MSC phenotype (CD105(pos)CD73(pos)CD45(neg)CD34(neg)), displayed multilineage differentiation potential, and suppressed alloreactivity in mixed lymphocyte reactions. V-BM may be an excellent source for MSC cotransplantation approaches.

Substance P (SP) induces endocytosis and recycling of the neurokinin 1 receptor (NK1R) in endothelial cells and spinal neurons at sites of inflammation and pain, and it is thus important to understand the mechanism and function of receptor trafficking. We investigated how the SP concentration affects NK1R trafficking and determined the role of Rab GTPases in trafficking. NK1R trafficking was markedly influenced by the SP concentration. High SP (10 nM) induced translocation of the NK1R and beta-arrestin 1 to perinuclear sorting endosomes containing Rab5a, where NK1R remained for >60 min. Low SP (1 nM) induced translocation of the NK1R to early endosomes located immediately beneath the plasma membrane that also contained Rab5a and beta-arrestin 1, followed by rapid recycling of the NK1R. Overexpression of Rab5a promoted NK1R translocation to perinuclear sorting endosomes, whereas the GTP binding-deficient mutant Rab5aS34N caused retention of the NK1R in superficial early endosomes. NK1R translocated from superficial early endosomes to recycling endosomes containing Rab4a and Rab11a, and Rab11aS25N inhibited NK1R recycling. Rapid NK1R recycling coincided with resensitization of SP-induced Ca2+ mobilization and with the return of surface SP binding sites. Resensitization was minimally affected by inhibition of vacuolar H(+)-ATPase and phosphatases but was markedly suppressed by disruption of Rab4a and Rab11a. Thus, whereas beta-arrestins mediate NK1R endocytosis, Rab5a regulates translocation between early and sorting endosomes, and Rab4a and Rab11a regulate trafficking through recycling endosomes. We have thus identified a new function of Rab5a as a control protein for directing concentration-dependent trafficking of the NK1R into different intracellular compartments and obtained evidence that Rab4a and Rab11a contribute to G-protein-coupled receptor recycling from early endosomes.

Tachykinins interact with three neurokinin receptors (NKRs) that are often coexpressed by the same cell. Cellular responses to tachykinins depend on the NKR subtype that is activated. We compared the colocalization of NK1R and NK3R with beta-arrestins 1 and 2, which play major roles in receptor desensitization, endocytosis, and signaling. In cells expressing NK1R, the selective agonist Sar-Met-substance P induced rapid translocation of beta-arrestins 1 and 2 from the cytosol to the plasma membrane and then endosomes, indicative of interaction with both isoforms. In contrast, the NK3R interacted transiently with only beta-arrestin 2 at the plasma membrane. Despite these differences, both NK1R and NK3R similarly desensitized, internalized, and activated MAP kinases. Because interactions with beta-arrestins can explain differences in the rate of receptor resensitization, we compared resensitization of agonist-induced Ca2+ mobilization. The NK1R resensitized greater than twofold more slowly than the NK3R. Replacement of intracellular loop 3 and the COOH tail of the NK1R with comparable domains of the NK3R diminished colocalization of the NK1R with beta-arrestin 1 and accelerated resensitization to that of the NK3R. Thus loop 3 and the COOH tail specify colocalization of the NK1R with beta-arrestin 1 and determine the rate of resensitization.

We evaluated the contribution of rab5a and rab11a to trafficking and signaling of protease-activated receptor 2 (PAR2), a receptor for trypsin and tryptase. Agonists stimulated internalization of PAR2 into early endosomes containing rab5a. Dominant negative rab5aS34N disrupted early endosomes and inhibited agonist-stimulated endocytosis of PAR2. Internalized PAR2 was sorted to lysosomes, and rab5a remained in early endosomes. Rab5a promoted and rab5aS34N impeded resensitization of trypsin-induced calcium mobilization. Rab11a was detected in the Golgi apparatus with PAR2, and PAR2 agonists stimulated redistribution of rab11a into vesicles containing PAR2 that migrated to the cell surface. Dominant negative rab11aS25N was mostly confined to the Golgi apparatus. Although expression of rab11aS25N caused retention of PAR2 in the Golgi apparatus, it did not abolish trafficking of PAR2 to the cell surface. However, expression of wild-type rab11a accelerated both recovery of PAR2 at the cell surface and resensitization of PAR2 signaling. Thus rab5a is required for PAR2 endocytosis and resensitization, whereas rab11a contributes to trafficking of PAR2 from the Golgi apparatus to the plasma membrane.

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.

Rat insulinoma 1046-38 cells represent a model system to study beta-cell function. The mRNAs for sst1 and sst2, two of the five somatostatin receptors, were detected by reverse transcription polymerase chain reaction amplification in these cells. Displacement binding analysis suggested that sstl represents the major somatostatin receptor subtype. The sstl selective compound CH-275 did not inhibit adenylyl cyclases while compounds that activated sst2 did. In contrast, CH-275 caused a marked inhibition of voltage-operated Ca2+ channels while the sst2 specific analog octreotide elicited a less pronounced effect suggesting that in rat insulinoma 1046-38 cells sst1 preferably mediates the inhibition of Ca2+ channels.

Endocytosis of somatostatin receptors could regulate cellular responses to the two natural peptides, somatostatin-14 and somatostatin-28, and to synthetic ligands used in the clinical diagnosis and symptomatic therapy of neuroendocrine tumours. The five cloned SSTRs with or without epitope tags at their carboxyl-termini were expressed in rat insulinoma 1046-38 cells. Application of the two natural peptides or octreotide, at 37 degrees C but not at 4 degrees C, to cells transfected with somatostatin receptor subtype 2 or 3 cDNA resulted in a significant decrease of cell surface binding-sites for 125I-Tyr11-somatostatin-14. In contrast, cells transfected with subtype 5 cDNA only responded to stimulation with octreotide or somatostatin-28. Cells transfected with subtype 1 cDNA responded to somatostatin-14 and 28, while cells expressing subtype 4 cDNA showed no response. Confocal microscopy revealed that 6 min after stimulation with somatostatin-14 at 37 degrees C, tagged somatostatin receptor subtypes 1, 2 and 3 were internalized into vesicles. Internalization was not observed at 4 degrees C in the presence of 0.4 M sucrose and 80 microM phenylarsine oxide and hence proceeded via endocytosis through clathrin-coated pits and vesicles. After 20 min the internalized receptors appeared in perinuclear vesicles and after 120 min they reappeared at the plasma membrane. This recycling was not sensitive to cycloheximide and, hence, not dependent on de novo protein synthesis. Recovery of cell surface receptors was, however, inhibited by brefeldin A, monensin and bafilomycin A1, indicating that receptor recycling proceeded through vesicular traffic of acidified compartments. The data are consistent with the assumption that the observed agonist and subtype specific internalization of somatostatin receptors in a neuroendocrine cell line may be important for tumour diagnosis and therapy and, thus, suggest a manifold control in cellular signalling.

Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.

We report the isolation and characterisation of cDNAs encoding three different, human members of the cysteine-rich secretory protein (CRISP) family. The novel CRISP-1 exists in five cDNA subtypes differing by the presence or absence of a stretch coding for a C-terminal cysteine-rich domain so far found in all members of the family, and by the length of their 3'-untranslated region. CRISP-2 cDNA corresponds to the previously described TPX1 form, with so far unreported 5'-untranslated sequence heterogeneities while CRISP-3 cDNA codes for a new, unique protein. Northern blot analysis of various human organs indicates that CRISP-1 transcripts are epididymis-specific whereas CRISP-2/TPX1 transcripts are detected mainly in the testis and also in the epididymis. CRISP-3 transcripts are more widely distributed and found predominantly in the salivary gland, pancreas and prostate, and in less abundance in the epididymis, ovary, thymus and colon. A protein reacting with an anti-mouse CRISP-1 antibody was isolated from human epididymal extracts and N-terminal sequencing revealed that it corresponded to the CRISP-1 cDNA we have isolated. In contrast to findings on its rat counterpart epididymal protein DE/acidic epididymal glycoprotein (AEG), no significant association of CRISP-1 with human spermatozoa was observed.

In the rat, the secretory glycoprotein DE/AEG is one of the main constituents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of approximately 6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented approximately 15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein.