Biomedical subjects

M Chorev

Publications and source records attributed to M Chorev.

At least 109 records · Page 6Linked to original sources

PubMed1990

Proteolytic resistance and biological activity of N-methylated analogs of [pGlu6] substance P6-11.

Five synthetic N-methylated analogs (II-V) of the C-terminal hexapeptide analog of substance P (SP), [pGlu6]SP6-11 (I) were evaluated for their metabolic stability and in vitro spasmogenic activity. The metabolic resistance of the analogs was tested by two SP degrading systems with different specificities, namely, the rat parotid and the hypothalamic slice systems. Their biological activity was assessed in the isolated guinea pig ileum. The analog [pGlu6, N-Me Phe7, N-Me Gly9]SP6-11 (III), had relative potency of 65% in the spasmogenic assay as compared to the parent compound. It was found to be more stable than the parent peptide in the hypothalamus, whereas in the parotid system it was susceptible as the parent peptide. However, the analog [pGlu6, N-Me Leu10]SP6-11 (II) (46% relative potency in the spasmogenic assay) was more stable than the parent peptide in the parotid system but did not show any improved stability in the hypothalamus. Identification of degradation products of the [pGlu6, N-Me Leu10]SP6-11 reflected the differences in the specificities of the two preparations. A significant drop in potency (7%) was observed for [pGlu, N-Me Phe7]SP6-11 (IV). This analog was more stable in the hypothalamic system than in the parotid. Introduction of a double methylation, [pGlu6, N-Me Leu10] SP6-11, contributed toward the stabilization in both degrading systems. Its relative spasmogenic activity was comparable to that of analog IV. In light of the above mentioned findings the implications of the N-methylated analogs with respect to putative CNS activity are discussed.

Animals↗

PubMed1990

Effects of hydrophobic substitutions at position 18 on the potency of parathyroid hormone antagonists.

Position 18 in a parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2 (ii), was shown to tolerate substitutions by a range of amino acids with retention of inhibitory activity. The effects of hydrophobic substitutions at this position as a means of enhancing binding interactions with the receptor were evaluated. Substitution of Nle at position 18 with either D-Ala, D-Trp, or L-Trp in analog ii or with Trp (D or L) in the recently reported, highly potent antagonist, [Nle8,18,D-Trp12,Tyr34]bPTH(7-34)NH2 (in vitro activities; Kb = 15 nM and Ki = 125 nM), was performed. In terms of activity on renal receptors, one antagonist, [Nle8,D-Trp12,18,Tyr34]bPTH(7-34)NH2, is the most active in vitro PTH antagonist yet reported (Kb = 4 nM; Ki = 30 nM). The rationale for design of this antagonist and the conclusions regarding PTH-receptor interactions are discussed.

Adenylyl Cyclase Inhibitors↗

PubMed1990

Downregulation of parathyroid hormone receptors in renal membranes from aged rats.

The mechanism of the inhibition or blunting of parathyroid hormone (PTH)-stimulated Na(+)-Ca2+ exchange activity in renal cortical cells from aged rats was examined. The number of PTH binding sites in basolateral membranes prepared from adult (6 mo) and old (24 mo) rats was quantitated by the binding of the synthetic analogue 125I-labeled [Nle8,18, Tyr34]bPTH-(1-34) amide to the membrane. The maximum number of specific PTH binding sites, Bmax, was 92.7 +/- 9.3 and 36.7 +/- 6.1 fmol/mg protein, respectively, in membranes prepared from adult and old rats. The affinity of the receptor to PTH was unaffected with age. The level of PTH binding components (68 and 70 kDa) estimated by a ligand affinity blot technique using biotinylated bPTH-(1-34) as the ligand was similarly reduced in membranes isolated from senescent rats. To test the hypothesis that change in the number of PTH binding sites and level of PTH binding components represented an adaptive response to a high serum PTH level, rats were parathyroidectomized (PTX) and the changes were reexamined. Decreases in the number of PTH binding sites and PTH binding components were either partially or completely negated by the surgery. These findings suggest that the blunting of both the PTH-stimulated Na(+)-Ca2+ exchange and adenylate cyclase activities in the kidneys of aged rats was due, in part, to be loss of PTH receptors in basolateral membranes and that this defect could be partially reversed by removal of the parathyroid gland.

Adenylyl Cyclases↗

PubMed1989

Substance P-induced cutaneous plasma extravasation in rats is mediated by NK-1 tachykinin receptors.

Substance P (SP), a relatively non-selective tachykinin receptor agonist, and Septide and Senktide, highly selective NK-1 and NK-3 tachykinin receptor agonists, respectively, were injected intradermally in rats. The resulting cutaneous plasma extravasation (PE) was evaluated by measuring the amount of Evans blue that leached from the circulation into the skin. SP and Septide produced dose dependent PE, Septide being the more potent of the two. Senktide did not produce PE, even at doses 10,000 times higher. Neonatal capsaicin treatment significantly reduced SP- and Septide-induced PE. These data indicate that SP-induced PE is mediated by NK-1 tachykinin receptors.

Animals↗

PubMed1989

1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6.

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.

Dimethyl Sulfoxide↗

PubMed1989

Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc-tyrosine aminomalonate, effectively block the IRK-catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti-lipolytic effect of the hormone. Only tBocTyr-aminomalonate was found to inhibit all the effects of insulin measured: insulin-stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin-dependent lipogenesis and the insulin-dependent anti-lipolytic effect. This inhibitor is the first blocker which is reported to block insulin-dependent anti-lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin-independent lipolysis, on [U14C]fructose assimilation or on (-)isoproterenol-stimulated lipolysis. These studies suggest that insulin-dependent lipogenesis and anti-lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.

Adipose Tissue↗

PubMed1988

The actions of receptor-selective substance P analogs on myenteric neurons: an electrophysiological investigation.

Intracellular recordings were made from myenteric neurons of the guinea-pig duodenum and the responses to local ejection of several substance P (SP) analogs were examined. It was found that senktide (succinyl-[Asp6,Me-Phe8]SP-(6-11)), a selective analog for the NK-3 (SP-N) receptor, was particularly effective in depolarizing the neurons. It was 20-100 times more potent than SP and about 1000-fold more potent than the selective analogs for the NK-1 (SP-P) receptor, which resides on muscle cells. The response to the peptides was prolonged (20-120 s), but in about 20% of the cells there was a fast, early depolarizing component (observed only with senktide). In most cases there was an increase in the input resistance of the cell during the slow depolarization. Together with the finding that the response reversed at about -90 mV, this indicates that the response is due to the closure of K+ channels. The results support the existence of an NK-3 (SP-N) receptor and provide direct information about the membrane mechanisms through which NK-3 agonists excite myenteric neurons.

Action Potentials↗

PubMed1988

Effect of substance P on rat gastrointestinal transit.

The in vivo effect of substance P and related peptide analogs on gastrointestinal transit in unanesthetized rats was studied. Fasted male rats were given intragastrically 0.5 ml of a powdered charcoal (BaSo4.H2O) meal and were concomitantly injected intraperitoneally with 8 micrograms/kg of substance P or a related peptide. In control rats, the percentage of small intestine traversed by the meal 15 min after feeding was 44.9 +/- 1.4 (N = 12). Substance P, [pGlu6]SP, [pGlu6, gPhe8, mGly9]SP and [pGlu5, N-MePhe8, N-MeGly9]SP significantly accelerated intestinal transit: 59.5 +/- 3.1% (N = 7); 66.0 +/- 3.8% (N = 14), 66.8 +/- 2.4% (N = 25), and 58.4 +/- 4.4% (N = 4), respectively. Concomitant injection of [pGlu6]SP and BOC-Phe-Phe-Gly-NHOH, an inhibitor of enzyme degradation at a dose of 800 micrograms/kg lowered by 10-fold the dose of [pGlu6]SP needed to induce the same degree of intestinal transit acceleration. These results indicate that in rats, substance P and related peptides accelerate gastrointestinal transit.

Animals↗

PubMed1988

Dehydro keto methylene and keto methylene analogues of substance P. Synthesis and biological activity.

The synthesis of dehydro keto methylene and keto methylene analogues of substance P using classical peptide synthesis is described. The following analogues were prepared: [pGlu6,Gly9psi(COCH2)(RS)Leu10]SP6-11 (4) and [pGlu6,-(RS)Phe7 psi(COCH2)(RS)Phe8]SP6-11 (8). The use of an improved deprotection scheme employing Meerwein's reagent (Et3OBF4) made possible the syntheses of the novel dehydro keto methylene analogue [pGlu6,(RS)Phe7 psi-(COCH2)delta(E)Phe8]SP6-11 (26) adn the tetrapeptide analogue [pGlu6,(RS)Phe8 psi(COCH2)Gly9]SP6-9(-OMe) (23). Compound 4 was a weak agonist in provoking contractions of the guinea pig ileum. Compound 26 was a potent inhibitor of SP degradation in rat hypothalamus preparations, with an IC50 value of 1.8 microM.

Animals↗

PubMed1988

Evaluation of novel parathyroid hormone analogs using a bovine renal membrane receptor binding assay.

A PTH membrane receptor binding assay based on a stable hormone analog radioligand was refined and used with bovine renal cortical membranes to evaluate PTH reference peptides and novel analogs of the hormone. Systematic studies were performed to optimize several aspects of the receptor binding assay. The sulfur-free agonist analog [Nle8,18,Tyr34]bovine PTH-(1-34)NH2 was iodinated using Iodogen. The monoiodinated derivative was purified and isolated by reverse phase HPLC. Immediate dilution of the purified radioligand in albumin-containing buffer and cold storage of aliquots yielded a tracer that was stable for at least 2 months. When used in the binding assay, the radioligand displayed specific, high affinity, and saturable binding to both bovine and canine renal cortical membranes. The Kd values were 0.47 +/- 0.07 and 0.63 +/- 0.08 nM for bovine and canine membranes, respectively. Maximum binding values were 475 +/- 46 and 395 +/- 48 fmol/mg protein for bovine and canine membranes, respectively. Furthermore, when used on a routine basis, this assay system proved reliable and reproducible. A series of previously characterized PTH agonists and antagonists was tested as reference peptides to validate this assay. Inhibition of binding by agents was dose dependent, parallel concentration-dependent binding curves were observed, and a close correlation between binding affinity and adenylate cyclase activity was obtained. In addition, a series of novel analogs designed to determine the consequences of sequence elongation at the N- and/or C-terminus or incorporation of amino acid substitutions to enhance resistance to enzymatic degradation were examined. With the exception of C-terminal extension from position 34 to position 38, which diminished receptor affinities, all modifications retained full biological activity. This refined receptor binding assay should facilitate future studies of newly designed PTH analogs.

Adenylyl Cyclases↗

PubMed1988

A new highly potent parathyroid hormone antagonist: [D-Trp12,Tyr34]bPTH-(7-34)NH2.

Based upon N-terminal parathyroid hormone (PTH) analog structure-activity relationship studies, position 12 was found to possess a wide structural latitude and was chosen as a site for single amino acid substitutions. Replacement of the naturally-occurring Gly with D-Trp at position 12 in the PTH antagonists [Tyr34]bPTH-(7-34)NH2 and [Nle8,18,Tyr34]bPTH-(7-34)NH2 increased in vitro receptor affinity. The D-Trp12 containing analogs were 12-fold more potent than their unsubstituted counterparts as inhibitors of PTH binding to renal and bone PTH receptors and 13-27-fold more potent as inhibitors of PTH-stimulated renal and bone adenylate cyclase activity. Based upon Scatchard analyses of saturation binding experiments and Schild analyses of adenylate cyclase experiments, [D-Trp12,Tyr34]bPTH-(7-34)NH2 was shown to interact with PTH receptors in a competitive manner. These studies demonstrate, therefore, that D-Trp12 substitution in PTH antagonists improves inhibitory properties in vitro and is compatible with a helical conformation at this position as a new direction for the design of PTH antagonists.

Adenylyl Cyclases↗

PubMed1988

Desensitization with a selective agonist discriminates between multiple tachykinin receptors.

We have reported recently the development of substance P analogs which selectively activate different tachykinin receptor types. These compounds, namely septide [( pGlu6, Pro9]substance P6-11 hexapeptide), which preferentially acts on the NK-1 (SP-P) receptor, and senktide (succinyl-[Asp6,Me-Phe8] substance P (6-11) hexapeptide), which selectively acts on the NK-3 (or SP-N) receptor, were now used to identify the tachykinin receptors present in several smooth muscle systems. Septide as well as senktide were found to be devoid of activity in the electrically stimulated rat vas deferens, showing that, in this preparation, which is the prototype NK-2 (or SP-E) system, tachykinin-induced contraction is not mediated via NK-1 or NK-3 receptors. In contrast, senktide did evoke contraction of the isolated rat duodenum, demonstrating that this tissue, which had been classified previously as a NK-2 preparation, also contains NK-3 receptors. After desensitization of NK-3 receptors by treatment with a desensitizing concentration of senktide, the rank order of tachykinin potencies in contracting the isolated rat duodenum corresponded to that of a typical NK-2 system. In the guinea pig ileum in which the tachykinin-evoked contraction is mediated by both NK-1 and NK-3 receptors, desensitization of NK-3 receptors unmasked a typical NK-1 response. Inasmuch as selective desensitization of both NK-1 and NK-3 sites abolished the tachykinin-induced response of the guinea pig ileum, it seems that this preparation does not contain NK-2 receptors. Our results show that selective agonists can be used to perform independent assays of several tachykinin receptors present within the same tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Metabolically stable analogues of substance P: persistent action of partially modified retro-inverso analogues of substance P on rat parotid and hypothalamic slices.

In a search for metabolically stable analogues of substance P (SP) the hexapeptide [pGlu6]SP-(6-11) was modified by reversal of the direction of a single amide bond. This novel peptide modification reverses the direction of the amide bonds at the peptide backbone but attempt to retain the topology of the amino acid side-chains at the peptide surface. The partial retro-inverso modification was successfully applied in a previous study for enkephalin analogues which were found to have potent and protracted morphinomimetic activity both in vivo and in vitro. The partially modified retro-inverso analogues: [pGlu6 psi(NH-CO)(RS)-Phe7]SP-(6-11) (analogue II) and [pGlu6,Phe8 psi(NH-CO)Gly9]SP-(6-11) (analogue III) were tested on guinea-pig ileum and for K+ release from rat parotid slices. Metabolic stability of the analogues was measured by their ability to produce persistent K+ release from parotid slices, their half life time (t1/2) in the rat parotid and hypothalamic slice systems and their resistance to proteolytic cleavage by chymotrypsin, pepsin, papain and pronase. Analogue II was devoid of biological activity and was slowly degraded in the parotid system and by several proteases. Analogue II was a full agonist of the SP-P receptor with a potency of 22 and 15% of the parent compound I, in the guinea-pig ileum and parotid slice system respectively. Pretreatment of the guinea-pig ileum with atropine (0.3 microM) had no effect on the potency of analogue III. On the other hand, when tested on rat vas deferens (an SP-E system), analogue III was about 20-fold more potent than the parent compound I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand.

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.

Animals↗

PubMed1986

Sequence determination of N-terminal and C-terminal blocked peptides containing N-alkylated amino acids and structure determination of these amino acid constituents by using fast-atom-bombardment/tandem mass spectrometry.

Peptides, blocked either at the N or C terminus, and thus unsuited for Edman degradation, and those containing N-alkylated amino acids, which are not detectable when using conventional amino acid analysis, can be easily sequenced by applying a method in which fast atom bombardment (FAB) is combined with tandem mass spectrometry (MSMS). Moreover, the structure of the N-alkylated amino acid constituents is provided by this approach. A widely applicable strategy will be presented, and to demonstrate its scope and limitations eighteen analogues of sequences related to the C terminus of substance P, a biologically active neuropeptide, were investigated. The power and reliability of the approach will be demonstrated by analyzing an 'unknown' peptide. Moreover, the detection and structure elucidation of N-alkylated amino acids which usually escape amino acid analysis will be described, as will be the unequivocal differentiation and identification of isomeric MeLeu/MeIle. The influence of the N-alkylation on the mass spectrometric fragmentation behaviour will be discussed. Furthermore, the sequencing of two adipokinetic hormones by using the combined FAB-MSMS approach is described. Analysis of peptides can be achieved with sample sizes less than 0.1 mumol and be completed within 2-4 h.

Alkylation↗

PubMed1986

Stereospecific antibodies to propranolol.

The beta-adrenergic antagonist propranolol was activated through its side chain, coupled to bovine serum albumin, and injected into BALB/c mice. After fusion of the splenocytes from these immunized mice with the NS-1 myeloma cell line, two hybridomas, producing monoclonal anti-propranolol antibodies, were isolated. Clone P-49 was monospecific for propranolol, with a significant preference for the 1-stereoisomer, as compared to the d form. On the other hand, clone P-28 cross-reacted with alprenolol as well as some other beta-antagonists. Both classes of antibodies competed with A431 epidermoid carcinoma beta 2-adrenoceptors for the binding of [3H]propranolol. When ascites cells from clone P-28 were fixed with glutaraldehyde, the anti-propranolol monoclonal antibody became cell bound. These cell-bound P-28 antibodies bind propranolol and other beta-adrenergic ligands with a similar ranking order to the soluble monoclonal antibody. The cell-bound antibody displayed a 5-fold higher affinity towards 1-propranolol than the soluble monoclonal antibody. The practical implications of these findings are discussed.

Antibodies, Monoclonal↗

PubMed1986

Association of turkey erythrocyte beta-adrenoceptors with a specific lipid component.

We have recently reported that the highly potent beta-adrenergic affinity label [125I]bromoacetylamino cyanopindolol ([125I]BAM-CYP) irreversibly blocks the turkey erythrocyte beta-adrenoceptor binding site by combining with a receptor-associated non-protein component. In this communication, we report: lipid labelling is inhibited by beta 1-adrenergic ligands with the potency ratio and stereospecificity characteristic for the turkey erythrocyte beta 1-adrenoceptor; the tagged component is a glycolipid, probably a ganglioside; [125I]BAM-CYP-blocked receptor, after solubilization in deoxycholate, can be separated from the [125I]BAM-CYP-glycolipid with restoration of the binding capacity of the beta 1-adrenoceptor protein; the tightly associated [125I]BAM-CYP-labelled glycolipid can be displaced by a glycolipid mixture extracted from turkey erythrocyte membranes but not by bovine brain gangliosides, when the blocked receptor is solubilized in digitonin. This is the first direct demonstration that a receptor protein is associated with a specific membrane lipid. The possibility that glycolipids play a role in receptor-mediated signal transduction is discussed in view of these findings and in view of data from the literature.

Affinity Labels↗

PubMed1986

Highly selective agonists for substance P receptor subtypes.

The existence of a third tachykinin receptor (SP-N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N-methylation of individual peptide bonds in the C-terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl-[Asp6,Me-Phe8]SP6-11, elicits half-maximal contraction of the guinea pig ileum through the neuronal SP-N receptor at a concentration of 0.5 nM. At least 60,000-fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP-P and SP-E). The action of selective SP-N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP-N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.

Acetylcholine↗