Specific binding of 3H-substance P to rat brain membranes

Hanley, Michael R.; Sandberg, Bengt; Lee, C. M.; Iversen, Leslie L.; Brundish, Derek E.; Wade, Roy

doi:10.1038/286810a0

Cited by 136 publications

(29 citation statements)

References 19 publications

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“…The C-terminal hexapeptide segment of substance P, 8b, administered intravenously to anesthetized rats has the same hypotensive activity as substance P. 7a In addition, the pentapeptide has been coupled to other pharmacologically active substances, 13,14 including an enkephalin active fragment, with retention of substance P activity. The pentapeptide itself was reported to be 100-and 2000-fold less potent than substance P in receptor binding 12 and contraction of guinea pig ileum, 4 respectively. Thus, SP 7-11 seemed a promising choice for the SP portion of the binary drug.…”

Section: Resultsmentioning

confidence: 99%

Binary drugs: conjugates of purines and a peptide that bind to both adenosine and substance P receptors

Jacobson¹,

Lipkowski²,

Moody³

et al. 1987

J. Med. Chem.

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A "functionalized congener" approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with K i values of 35 nM (central A 1 -adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N 6 -[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine.A "functionalized congener" approach to adenosine receptor antagonists 1,2 has resulted in highly potent 1,3-dialkylxanthines having amino and carboxylic acid functionalized chains. These functional groups have been coupled covalently to amino acids and dipeptides. The resulting amino acid and oligopeptide conjugates have full, and in some cases enhanced, biological activity. In our previous work 2 we used nonhormonal peptide sequences to alter the physicochemical properties of the drugs. We now report the attachment of a xanthine to a segment derived from a putative neurotransmitter peptide, substance P (SP) (reviews, ref 3,4), to form a binary drug 1 (Figure 1). This drug binds both to adenosine receptors and to substance P receptors with affinity comparable to that of each component at the appropriate receptor. In addition, coupling of the adenosine agonist N 6 -[p-(carboxymethyl)phenyl]adenosine, 5 2a, to substance P segment 7-11 produced the binary drug 3, which also binds to both receptors. We have demonstrated a potentially general strategy via "functionalized congeners" for the synthesis of binary drugs that combine pharmacological properties of both elements. The choice of combination of adenosine receptor ligands and substance P in target molecules stems from the peripheral hypotensive effect elicited by agonists for both receptor. 6,7 Moreover, since both receptors mediate central nociceptive effect, 8,9 the binary conjugates may prove to be useful tools in probing neuronal pathways involved in perception of pain. ResultsThe xanthine amine congener (XAC) has several features in its structure that lead to enhanced receptor affinities. Thus, we have found from a study of many xanthine derivatives that an amino group in the appended chain...

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Section: Resultsmentioning

confidence: 99%

Binary drugs: conjugates of purines and a peptide that bind to both adenosine and substance P receptors

Jacobson¹,

Lipkowski²,

Moody³

et al. 1987

J. Med. Chem.

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“…In any case, caution is needed in interpreting the relative K D values thus obtained, since the reverse-phase chromatography was carried out in 25 CH3CN and at low pH. The relative binding potency was determined by the ratios of (1'250 of substance P)/(ICso of analogue) in competing against the specific binding of 2 nM [3H] substance P to rat brain membranes as described previously [7]. The 1C50 of substance P was 3nM.…”

Section: Summary Of Physicochemical Immunological and Biological Pmentioning

confidence: 99%

“…Analogues were tested as inhibitors of the specific binding of [3H]substance P to a washed membrane fraction from rat brain as described previously [7]. Specific binding was defined by subtracting non-specific binding (determined in the presence of 10 pM substance P) from total binding.…”

Section: Substance P Binding Studies On Rat Brain Membrane Preparationmentioning

confidence: 99%

“…As described in the preceding paper [3], we have isolated an endopeptidase from human brain with a high specificity and affinity for substance P which cleaves the bonds Gln6-Phe7, Phe7-Phe8 and Phe8-Gly9. Any of these cleavages leads directly to inactivation of substance P. This information provided us with a rational basis for the design and synthesis of enzyme-resistant analogues of substance P. If such enzyme-resistant analogues were to be useful as pharmacological tools, they should mimic the biological activity and specificity of substance P. The synthetic analogues of substance P were, therefore, tested for their ability to contract the guinea pig ileum and to compete for specific binding of [3H]substance P to receptor sites in rat brain membranes [7]. The undecapeptides and the heptapeptides described in this paper were synthesized by the solid-phase method.…”

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confidence: 99%

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Synthesis and Biological Properties of Enzyme‐Resistant Analogues of Substance P

Sandberg

Lee

Hanley

et al. 1981

European Journal of Biochemistry

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Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-Pdegrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5 -11, < Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide.Fluorenylmethyloxycarbonyl was used as an a-amino protecting group in conjunction with t-butyloxycarbonyl as an eamino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ionexchanger. The peptides were finally purified on a semi-preparative reverse-phase column.The undecapeptide substance P (Fig. 1) has a number of peripheral actions which include contraction of smooth muscle, reduction of blood pressure and stimulation of secretory tissues. Like many other gut peptides, substance P also occurs in neurones in the central nervous system [1,2]. Substance P is of particular interest as a possible neurotransmitter or neuromodulator, released by primary sensory neurones [ 2 ] . Substance P is concentrated in the terminals of sensory neurones synapsing in the dorsal horn of the spinal cord, an area where it may be involved in transmitting pain sensation [2]. The occurrence of substance P in the central nervous system and the proposal that it may act as a neurotransmitter have focused interest on its metabolism. Rapid enzymatic inactivation is probably of importance in terminating the actions of the synaptically released peptide (see preceding paper [3]). The development of enzyme-resistant enkephalin analogues has contributed significantly to the understanding of enkephalin action and provides an avenue for the future design of peptide drugs [4]. To date, however, no metabolically stable analo...

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“…84 SUBSTANCE P ANAL YSIS IN THE BOVINE RETINA Uptake experiments. Fresh retinas were placed in Krebs bicarbonate medium containing the peptidase inhibitors bacitracin (300 psg/ml), phenylmethylsulphonyl fluoride (1 mM) and pchloromercuribenzoate (01 mM) (Hanley, Sandberg, Lee, Iversen, Brundish & Wade, 1980). After a pre-incubation for 10 min at 37 0C, [3H]substance P ([4-3H-Phe8]substance P from Cambridge Research Biochemicals specific activity 17-45 Ci/mmol) at a concentration of 1 UM was added and the incubation continued for 10-15 min.…”

Section: Introductionmentioning

confidence: 99%

Substance P in the bovine retina: localization, identification, release, uptake and receptor analysis.

Osborne¹

1984

The Journal of Physiology

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SUMMARY1. Substance P-like immunoreactivity is restricted to at least three different cell types in the bovine retina. They are amacrine cells, displaced amacrine cells and interplexiform neurones.2. High performance liquid chromatography and radioimmunoassay have shown that the bovine retina substance P-like immunoreactive material cannot be distinguished from authentic substance P.3. Isolated bovine retina does not take up exogenous [3H]substance P as revealed by autoradiography.4. By increasing the external K+ concentration it was shown that the release of endogenous substance P from the retina was stimulated. This release is Ca2+ dependent.5. The binding of [3H]substance P to crude membrane preparations of bovine retina was saturable and revealed a single population of binding sites with a KD value of 0-32 nm and a Bmax of 2x45 pmol/g wet wt. The characteristics of the binding site suggest the presence of substance P receptors.6. It is suggested that substance P is a likely neurotransmitter in the bovine retina.

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Specific binding of 3H-substance P to rat brain membranes

Cited by 136 publications

References 19 publications

Binary drugs: conjugates of purines and a peptide that bind to both adenosine and substance P receptors

Binary drugs: conjugates of purines and a peptide that bind to both adenosine and substance P receptors

Synthesis and Biological Properties of Enzyme‐Resistant Analogues of Substance P

Substance P in the bovine retina: localization, identification, release, uptake and receptor analysis.

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