Transferrin receptor containing the SDYQRL motif of TGN38 causes a reorganization of the recycling compartment but is not targeted to the TGN.

The SDYQRL motif of the cytoplasmic domain of TGN38 is involved in targeting TGN38 from endosomes to the TGN. To create a system for studying this pathway, we replaced the native transferrin receptor (TR) internalization motif (YTRF) with the SDYQRL TGN-targeting motif. The advantages of using TR as a reporter molecule include the ability to monitor trafficking, in both biochemical and microscopy experiments, using the natural ligand transferrin. When expressed in CHO cells, the SDYQRL-TR construct accumulated in juxtanuclear tubules and vesicles that are in the vicinity of the TGN. The SDYQRL-TR-containing structures, however, do not colocalize with TGN markers (e.g., NBD ceramide), and therefore the SDYQRL motif is not sufficient to target the TR to the TGN. The morphology of the SDYQRL-TR-containing juxtanuclear structures is different from the recycling compartment found in cells expressing the wild-type TR. In addition, the SDYQRL-TR-containing juxtanuclear compartment is more acidic than the recycling compartment in cells expressing the wild-type TR. The juxtanuclear compartment, however, is a bona fide recycling compartment since SDYQRL-TR was recycled back to the cell surface at a rate comparable to the wild-type TR, and sphingomyelin and cellubrevin, both of which label all compartments of the endocytic recycling pathway, colocalize with SDYQRL-TR in the juxtanuclear structures. These findings demonstrate that expression of the SDYQRL-TR construct alters the morphology and pH of endocytic recycling compartments rather than selectively affecting the intracellular trafficking pathway of the SDYQRL-TR construct. Therefore, the SDYQRL trafficking motif is not simply a molecular address that targets proteins to the TGN, but it can play an active role in determining the physical characteristics of endosomal compartments.

The carboxyl terminus of GLUT4 contains a serine-leucine-leucine sequence that functions as a potent internalization motif in Chinese hamster ovary cells.

To characterize the trafficking motifs contained in the carboxyl terminus of GLUT4, a chimera (GTCTR) was constructed in which the carboxyl-terminal 30 amino acids of GLUT4 were substituted for the amino-terminal cytoplasmic domain of the transferrin receptor (TR). The endocytic behavior of this chimera was characterized in Chinese hamster ovary cells. The GTCTR chimera had a more predominant intracellular distribution compared to the TR. Only 20% of the GTCTR chimera is on the surface at steady-state compared to 35% of the TR. The GTCTR chimera is internalized 50% more rapidly and recycled 20% more slowly than the TR. Acidification of the cytosol inhibited internalization of the GTCTR chimera, indicating that the chimera is internalized through clathrin-coated pits. Mutations of GTCTR were constructed in which a di-leucine sequence of the carboxyl domain of GLUT4 was mutated to a di-alanine sequence (GTCTR-AA) and serine residue 488, immediately preceding the di-leucine sequence, was mutated to either an alanine or aspartate residue. In each case, albeit to varying degrees, the substitutions shifted the distribution of the mutated GTCTR constructs toward the surface. The shift in the distribution of GTCTR-AA resulted from a 10-fold reduction in internalization, and the shift of serine 488 mutants resulted from a 3-fold reduction in the internalization rate compared to GTCTR. None of these mutations affected the recycling rate. These results demonstrate that the carboxyl terminus of GLUT4 contains a serine-leucine-leucine-based motif that, when expressed in non-insulin responsive cells, functions as a potent internalization motif which promotes more rapid internalization than does the native TR internalization motif.

Comparison of cyclic and linear analogs of vasoactive intestinal peptide.

A series of i-->i + 4 side-chain to side-chain lactam analogs of vasoactive intestinal peptide has been prepared in order to study the effect of cyclization on biological activity. In vitro, on guinea pig tracheal smooth muscle and on human bronchial tissue, approximately half of the cyclic analogs showed increased potency and half were decreased over the linear analogs. Several cyclic compounds were between 10- and 20-fold more potent and one was 290-fold more potent than the linear species. In vivo, in guinea pigs, the cyclic compounds showed increased potency by up to 70-fold and significantly enhanced duration of action as compared to linear compounds.

Structure-activity studies on the vasoactive intestinal peptide pharmacophore. 1. Analogs of tyrosine.

From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc-substituted-phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac-[Lys12,Nle17,X22,Val26,Thr28]-VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up to 300-fold. A few analogs, all possessing electron-donating substituents, retained nearly full potency. Two compounds, 3-F,4-OH-Phe, 42 and 3-OCH3,4-OH-Phe, 43, were found to be 1.5- and 3.4-fold more potent than the parent compound, which equates to being 8.9- and 20-fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2-fold enhanced potency and a significantly longer duration of action (> 20 min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part II: Effect on in vitro and in vivo models of pulmonary anaphylaxis.

Studies were conducted to compare the effect of native vasoactive intestinal peptide (VIP), Ro 25-1553 (a cyclic peptide analog of VIP) and salbutamol (a beta2-adrenoceptor agonist) on antigen-induced pathophysiological effects in the guinea pig. Ro 25-1553 and salbutamol (0.01-1.0 microM) prevented antigen-induced contractions of the guinea pig trachea in vitro with IC50 values of 0.07 and 0.05 microM, respectively. VIP (0.01-1.0 microM) had no effect on antigen-induced tracheal contractions. Aerosolized Ro 25-1553 and salbutamol were equipotent in preventing antigen-induced increases in guinea pig lung resistance (IC50 value = 0.0001%), whereas aerosolized VIP (0.1%) was ineffective. Ro 25-1553 (0.1-100 micrograms), instilled intratracheally 2 min before the antigen challenge of buffer-perfused lungs from sensitized guinea pigs, produced a dose-dependent inhibition of bronchoconstrictor, vasoconstrictor and edemagenic responses, whereas intratracheal VIP (100 micrograms) had no effect. Intratracheal salbutamol (0.1-100 micrograms) inhibited antigen-induced responses in a manner comparable to Ro 25-1553. Lung inflammation was assessed as leukocyte accumulation in bronchoalveolar lavage fluid after the antigen provocation. Aerosolized antigen-induced bronchoalveolar lavage eosinophilia (13-fold increase over saline controls) at 6 hr after challenge was prevented in a concentration-dependent manner by pretreatment with nebulized Ro 25-1553 and salbutamol, but not by pretreatment with native VIP. These results indicate that Ro 25-1553 suppresses various pathophysiological features associated with pulmonary anaphylaxis and asthma, including airway reactivity, edema formation and granulocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part I: In vitro and in vivo bronchodilator studies.

Ro 25-1553, a cyclic peptide analog of vasoactive intestinal peptide (VIP), was designed to overcome many of the deficiencies inherent in this natural neuropeptide. On isolated guinea pig tracheal smooth muscle, Ro 25-1553 produces concentration-dependent relaxation of contractile responses to a number of different spasmogens. Depending on the contractile stimulus, Ro 25-1553 is 24 to 89 times more potent than VIP as a relaxant of guinea pig trachea. The high potency of Ro 25-1553 extends to studies on isolated, histamine-contracted, human bronchial smooth muscle, where Ro 25-1553 exhibits a 390-fold enhancement over native VIP and is more potent than other bronchodilating drugs, such as the beta 2-adrenoceptor agonists isoproterenol and salbutamol. Ro 25-1553 was shown to displace the radioligand 125I-VIP from rat forebrain membranes with an IC50 value of 4.98 nM, thereby demonstrating that it acts at a VIP receptor. In addition, when tested in a battery of 40 other binding assays (e.g., muscarinic, histamine, LTs, Ca++, TxA2, endothelin, alpha and beta adrenergic, platelet-activating factor, neurokinins, etc.) at concentrations as high as 10 microM, Ro 25-1553 was found to be inactive; thus it appears to be specific for VIP receptors. The potent smooth muscle relaxant activity exhibited in vitro by Ro 25-1553 is also evident after in vivo intratracheal administration or aerosolization of the compound. Pulmonary responses evoked by histamine, leukotriene D4, platelet-activating factor and acetylcholine are inhibited dose-dependently by intratracheally instilled Ro 25-1553 with nearly identical potency (ED50 values ranging from 0.07 micrograms to 0.26 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain.

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs.

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

Structure-activity studies of vasoactive intestinal polypeptide.

This report explores the potential side-chain functional groups required for interaction of the bronchodilator neuropeptide, vasoactive intestinal peptide (VIP), with its receptor. The binding affinity and biological activity of native VIP have been found to be sensitive to the removal of amino- and carboxyl-terminal residues. This data suggests that elements within the entire primary sequence of the VIP molecule appear to be necessary for recognition by VIP receptors. The introduction of alanine residues substituted into the VIP molecule is utilized to probe for side-chain functional groups that are crucial for eliciting high receptor binding affinity in vitro and high biological potency in vivo. The VIP pharmacophore appears to be identical in guinea pig lung and human lung and consists of multiple binding sites most likely involving positions Asp3, Phe6, Thr7, Tyr10, Tyr22, and Leu23. These findings could be exploited to enhance the biological potency of VIP by increasing the binding energy at these positions.

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle.

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.

Mechanisms of methacholine-induced coronary vasospasm in an experimental model of variant angina in the anesthetized rat.

Methacholine (Mch), when injected near the ostia of the coronary arteries, induces an intense coronary vasospasm which can be measured by the degree of S wave elevation monitored from an electrocardiogram (ECG). The observed ECG changes resemble those occurring in patients with variant angina. The effects of Mch were blocked by atropine, but not by d-tubocurarine, hexamethonium, adrenergic receptor blockade, or prior reserpinization, indicating that Mch is acting directly on muscarinic receptors to produce a vasoconstriction of the coronary arteries. This model of Mch-induced coronary vasospasm appears to be useful for testing spasmolytic agents which might be of benefit in variant angina.