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Biomedical subjects

E Escher

Publications and source records attributed to E Escher.

At least 73 records · Page 4Linked to original sources
PubMed1990

Photoaffinity labelling of atrial natriuretic factor (ANF)-R1 receptor by underivatized 125I-ANF. Involvement of lipid peroxidation.

In bovine adrenal zona glomerulosa, atrial natriuretic factor (ANF) exerts its physiological effect through high-affinity binding to specific membrane receptors. On studying further the molecular properties of the ANF receptor binding domain, we have observed that incubation of intact or solubilized bovine adrenal zona glomerulosa membranes with 125I-ANF-(99-126) followed by u.v. irradiation results in the irreversible labelling of a 130 kDa protein corresponding to the ANF-RI receptor. This process is time-, protein- and 125I-ANF-dependent. The apparently covalent nature of this complex is documented by its resistance to heat, guanidine hydrochloride, urea and trichloroacetic acid denaturation. Photolabelling with underivatized 125I-ANF is much more efficient with the ANF-R1 than with the ANF-R2 receptor. After photolysis, the covalently linked 125I-ANF is still sensitive to digestion by carboxypeptidase A, suggesting that ANF is linked by its N-terminal end to the receptor upon u.v. irradiation and that its C-terminal end is still freely accessible. Aerobic conditions and lipids are required for the photolabelling, suggesting a role in this process for malondialdehyde, a highly reactive secondary product associated with u.v.-induced lipid peroxidation. This simple method should provide a powerful tool in the accurate characterization of the hormone-binding domain of the ANF receptor.

Affinity Labels↗
PubMed1990

Visualization of neurokinin-3 receptor sites in rat brain using the highly selective ligand [3H]senktide.

The autoradiographic distribution of the neurokinin (NK)-3 receptor sub-type was visualized in the rat brain using [3H]senktide, a highly selective ligand, [3H]Senktide apparently binds to a single class of high affinity (Kd = 2.8 +/- 1.0 nM), low capacity (Bmax = 31.2 +/- 3.0 fmol/mg protein) sites in rat brain cortex. The ligand selectivity pattern reveals that eledoisin and senktide are potent competitors of both [3H]senktide and [125I]Bolton-Hunter eledoisin binding sites demonstrating the NK-3 nature of these sites. Autoradiographic data show that [3H]senktide binding sites are concentrated in mid-cortical layers, supraoptic nucleus, zona incerta, basolateral nucleus of the amygdala and interpeduncular nucleus. Much lower densities of binding are seen in most other areas such as the caudate-putamen and cerebellum. This distribution is similar, but not identical, to that previously reported for NK-3 sites using less selective ligands. It is most likely because less selective probes also bind to other classes of NK receptors. The higher selectivity of [3H]senktide is thus an important advantage for the precise characterization of NK-3 receptor binding parameters.

Animals↗
PubMed1990

gamma-Preprotachykinin-(72-92)-peptide amide: an endogenous preprotachykinin I gene-derived peptide that preferentially binds to neurokinin-2 receptors.

The presence of N-terminally extended forms of neurokinin A has recently been reported in the mammalian brain. Among them, gamma-preprotachykinin-(72-92)-peptide amide [gamma-PPT-(72-92)-NH2], a peptide derived by posttranslational processing of gamma-preprotachykinin, is most prominent. We report here that this peptide most likely acts on neurokinin-2 receptor sites since neurokinin A (a putative neurokinin-2 agonist) and gamma-PPT-(72-92)-NH2 are potent competitors of 125I-labeled gamma-PPT-(72-92)-NH2 binding whereas selective neurokinin-1 and -3 agonists are not. Moreover, the distribution of 125I-labeled gamma-PPT-(72-92)-NH2 and 125I-labeled neurokinin A binding sites are very similar in rat brain. On the other hand, 125I-labeled Bolton-Hunter-substance P (a neurokinin-1 ligand) and 125I-labeled Bolton-Hunter-eledoisin (a neurokinin-3 ligand) binding sites are differentially located in this tissue. Thus, it appears that gamma-PPT-(72-92)-NH2 binds to neurokinin-2 receptors and should be considered as a putative endogenous ligand for this receptor class.

Animals↗
PubMed1990

An angiotensin with prolonged action and blood pressure--lowering properties.

[Sar1,Phe(Br5)8] angiotensin II (Br5Ang II) is a specific, quasi-irreversible antagonist of angiotensin II (Ang II) in vitro. In vivo, this compound is a very potent, Ang II-specific antagonist with a very long duration of action against Ang II-induced blood pressure (BP) increases. In the "low-sodium" dog, this compound induces a prolonged BP reduction during and after intravenous infusion at doses comparable to cilazapril, a potent ACE inhibitor. The physicochemical and pharmacokinetic behavior of this peptide was therefore assessed to understand and interpret the prolonged antagonistic and antihypertensive activity of this peptide. Binding studies using beef adrenocortical membranes indicated specific binding of Br5Ang and related analogues to Ang II receptors with a Kd of 1.41 x 10(-9) M against iodinated [Sar1, D-Phe8]Ang II, a standard radioligand antagonist. Iodinated Br5AngII exhibited a very high degree of nonspecific binding to the membranes. It had an octanol-water partitioning coefficient of log P of + 0.903, a coefficient 84-fold higher than for [125I][Sar1, D-Phe8]Ang II. Association kinetics of [125I]Br5Ang II were similar to the standard ligand [125I] [Sar1, D-Phe8]Ang II, but the half-life of dissociation was four times higher (60 vs. 15 min at 20 degrees C). Molecular modeling indicates a practically identical conformational behavior of both peptides, Br5Ang II and [Sar1, D-Phe8]Ang II but with an expanded hydration shell over the Br5 residue. It is concluded that the prolonged duration of action is due to the increased hydrophobicity of the peptide, which leads to a slow dissociation from the Ang II receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1989

An angiotensin II antagonist with strongly prolonged action.

A highly hydrophobic analogue of angiotensin II (AT), [Sar1,(2',3',4',5',6'-Br5)Phe8]AT exhibited strong and persistent specific antagonism against AT, both in vitro and in vivo. This peptide exhibited 32% of the binding affinity of [Sar1]AT towards membranes of bovine adrenal cortex, it was a specific AT antagonist of irreversible character on smooth muscle assays, and it also suppressed for over 120 min at 7.10(-8) M/kg the blood pressure response towards AT in the rat blood pressure assay. This compound harbours therefore the potential of a new class of AT-specific antihypotensive drugs.

Adrenal Cortex↗
PubMed1989

Inhibition of angiotensinogen production by angiotensin II analogues in human hepatoma cell line.

The aim of this study was to examine in Hep G2, a human hepatoma-derived cell line, the presence of angiotensin II (ANG II) receptors and the effect of ANG II and its analogues on angiotensinogen production. The presence of ANG II receptors was demonstrated using a long-acting ANG II analogue, 125I-labeled [Sar1]ANG II. A single class of specific binding sites was identified in these cells with a dissociation constant (Kd) of 2 nM. The number and affinity of these binding sites were not changed by [Sar1]ANG II treatment over 24 h. ANG II showed an inhibitory effect on angiotensinogen production. [Sar1]ANG II also exhibited a similar inhibitory effect as that of ANG II but to a greater extent and therefore was used throughout these studies. [Sar1]ANG II inhibited angiotensinogen production in a dose-dependent manner, exhibiting a half-maximal inhibitory concentration (IC50) of 2 nM. Other ANG II analogues showed similar effects on angiotensinogen production. In order of decreasing ability, they were [Sar1]ANG II greater than [Sar1-Ala8]ANG II greater than [Sar1-Val8]ANG II greater than [Sar1-Val5-(Br5)-Phe8]ANG II greater than [Sar1-Val5-DPhe8]ANG II. Results of these studies show that the Hep G2 cell possesses specific ANG II receptors and that [Sar1]ANG II induces a dose-dependent inhibition of angiotensinogen production in this system.

Angiotensin II↗
PubMed1989

Ca2+, but not membrane lipid hydrolysis, mediates human chorionic gonadotropin production by luteinizing hormone-releasing hormone in human term placenta.

We postulated a role for lipid metabolism and Ca2+ in the LHRH-induced release of hCG by human placentas. Term placental cells in suspension prelabeled with [3H]myoinositol were stimulated without or with increasing concentrations of LHRH in the presence of 10 mM LiCl, and total inositol phosphate (IP) was measured by ion exchange chromatography; a nonsignificant 0.9 +/- 0.08-fold increase over the control value was observed. In contrast, placental cells stimulated with equimolar concentrations of angiotensin II (AII) induced a 4.6 +/- 0.9-fold increase in total IP (P less than 0.01), while rat pituitary cells showed 1.9 +/- 0.2- and a 2.4 +/- 0.07-fold increases in total IP production after stimulation with LHRH or AII, respectively (P less than 0.05). These increases were blocked by coincubation with specific LHRH and AII antagonists. When 1 x 10(6) placental cells were incubated with 45Ca2+ without and with increasing doses of LHRH for 0-75 s and then filtered under negative pressure, we observed significant incorporation of 45Ca2+. This influx was linear with incubation time, significantly more pronounced in cells exposed to LHRH than in control cells, and showed a dose-response curve to LHRH that reached maximal influx rates of 3.6 +/- 0.3 nM/min.1 x 10(6) cells with 10(-5) M LHRH. This response was completely blocked by coincubation with 10(-5) M LHRH antagonists; cobalt chloride and verapamil reduced it by 60% and 80%, respectively. Compared to placental cells stimulated with LHRH alone, those coincubated with LHRH and specific LHRH or Ca2+ antagonists released from 10-100% less hCG. We conclude that Ca2+ participates in the LHRH action in human placentas, but uncoupled to PI turnover.

Angiotensin II↗
PubMed1988

Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex.

The autoradiographic distribution of the 3 neurokinin (NK) receptor sub-types, NK-1, NK-2 and NK-3, was compared in rat cerebral cortex during post-natal development using [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands. Throughout brain development, NK-1 receptor sites are present in low densities with some enrichment seen in lamina III while NK-3 binding sites are concentrated in layers IV and V. However, it appears that NK-2 receptors are mostly expressed in lamina VI and only during the first two postnatal weeks. These results demonstrate further the existence and differential ontogeny of 3 classes of NK receptors in rat brain cortex.

Aging↗
PubMed1988

Synthesis and immunological evaluation of N-terminal, noncrossreactive tachykinin antigens.

The N-terminal hexa- or pentapeptide sequences of the three mammalian tachykinins substance P, neurokinin A, and neurokinin B have been synthesized by the conventional solid-phase procedure with 6-aminocaproyl-S-(acetamidomethyl)cysteine as a C-terminal spacer and attachment function. A fourth sequence, with an additional N-terminal 6-aminocaproyl residue on the substance P-hapten sequence, was cyclized N- to C-terminally. For this purpose, a four-level protection scheme has been applied: BOC-TFA for N-terminal protection and cleavage; TFA-stable but HF-labile anchoring function and side-chain protection; S-acetamidomethyl for semipermanent thiol protection. The side chain amino function of Lys was protected with NO2Z, stable against HF but readily cleaved with hydrogenation. The hapten sequences were coupled to maleimidated BSA, after the Acm group was removed by mercury/hydrogen sulfide treatment. Mice immunized with the three linear hapten sequences produced sera that were specific in enzyme-linked immunosorbant assay for the presented hapten and the respective tachykinin but displayed no crossreactivity at all toward the other haptens nor to one of the other tachykinins. It is concluded that this approach produced antisera, specific and selective for its respective mammalian tachykinins.

Animals↗
PubMed1988

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Affinity Labels↗
PubMed1987

Apparent molecular weight of substance P/neurokinin-1 receptors determined using a photoaffinity labelled probe, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P.

A photoreactive derivative, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P (SP) was prepared and iodinated using carrier-free [125I] to determine the apparent molecular weight of one sub-type of neurokinin (NK) receptor, the SP/NK-1 type. The unlabelled analogue competed for [3H]-SP sites with an IC50 of 10 nM. The radioactive photoprobe (KD approximately 0.17 nM, Bmax = 15.6 fmol/mg protein) was used to photoaffinity label membranes prepared from rat brain. Autoradiographs revealed that a single band with an apparent molecular weight of 46,000 daltons was specifically labelled. This labelling was inhibited by non-radioactive SP in a concentration-dependent manner (1.0 nM-0.1 mM) suggesting that the observed labelling represents the SP/NK-1 receptor type.

Affinity Labels↗
PubMed1987

Effects of substance P analogues in the rat tail-flick test.

Five substance P (SP) analogues which antagonize tachykinins on isolated organs were studied using the tail-flick test. None antagonized the nociceptive effect of 1.9 nmol of physalaemin when studied at the concentrations of 6.5 and 65.0 nmol. [D-alpha Npa7,9,10]SP (65.0 nmol) produced a flaccid paralysis of the hindlimbs and the tail in 2 out of 9 rats; however, [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP-(4-11) (6.5 nmol) produced this type of paralysis in 4 out of 6 rats so treated. The [D-Trp]SP analogue appears to be the most toxic. The results also suggest that the receptor for physalaemin in the spinal cord is different from that in peripheral tissues.

Animals↗
PubMed1987

Spinal actions of substance P analogues on cardiovascular responses in the rat: a structure-activity analysis.

Ten substance P (SP) analogues were tested for their effects on mean arterial pressure and heart rate following intrathecal administration in the pentobarbital anaesthetized rat. The 10 analogues are [D-Pro4,D-alpha Npa7,9,10]SP(4-11) (A-I), (D-alpha Npa7,9,10]SP (A-II), [D-Trp7,9,10]SP (A-III), [D-Pro4,D-Npa7,9,Phe11]SP(4-11) (A-IV), [D-Pro4,D-beta Npa7,D-alpha Npa9,D-Phe11]SP(4-11) (A-V), [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP(4-11) (A-VI), [D-Pro4,D-Trp7,9,10,Phe11]SP(4-11) (A-VII), [D-Pro4,D-Trp7,9,10,Trp11]SP(4-11) (A-VIII), [D-Trp7,9,10,Trp11]SP (A-IX), and [D-Pro4,D-Phe7,9,10,Phe11]SP(4-11) (A-X). At 6.5 nmol, the analogues containing the amino acid D-Npa (A-I, A-II, A-IV, and A-V) or D-Phe (A-X) in positions 7, 9, or 10 of SP or its C-terminal octapeptide are devoid of the long-lasting cardio- and vaso-depressor effects, which are otherwise seen with analogues containing the amino acid D-Trp (A-III, A-VI, A-VII, A-VIII, and A-IX) in the same positions. Some of the analogues containing D-Npa maintain the initial hypotensive effect seen with SP while the analogue containing D-Phe produces only a small hypertensive response. The 10 analogues when tested at a dose that failed to alter basal mean arterial pressure and heart rate did not block the cardiovascular responses elicited by SP and no cross desensitization was observed between SP and these analogues. It appears that these SP analogues exert cardiovascular effects in the rat spinal cord probably without interacting with SP receptors.

Animals↗
PubMed1986

Role of calcium in stimulus-secretion coupling on isolated frog interrenal gland.

The influence of extracellular calcium concentration on the steroidogenic response to ACTH and to the angiotensin II analogue [Sar1-Val5]AII has been studied in the frog, using a perfusion system technique. The release of corticosterone and aldosterone in the effluent medium was measured by specific radioimmunoassays. In calcium-free medium the stimulatory effect of ACTH (10(-9) M) was completely abolished whereas the response to dbcAMP (5 mM) was unchanged indicating that the role of calcium takes place before the formation of cAMP. Conversely, in the absence of calcium, angiotensin II (10(-7) M) was still able to stimulate corticosterone and aldosterone production. Addition of Co2+ (4 mM), a calcium antagonist, to the perfusion medium, inhibited partially the response of adrenal tissue to ACTH, dbcAMP and angiotensin. The voltage-dependent calcium channel blocker verapamil (10(-6) induced a dose-related inhibition of the corticotropic effect of ACTH. At the higher dose (10(-4) M), verapamil totally inhibited the stimulation of corticosterone and aldosterone production induced by ACTH. By contrast, at the same dose it did not alter the stimulatory effect of forskolin (2.4 X 10(-7)M) on corticosterone output, but significantly diminished forskolin-induced aldosterone response. Similarly, angiotensin-stimulated corticosterone production was slightly inhibited by 10(-4) M verapamil, whereas aldosterone response to angiotensin was totally abolished, indicating that verapamil may act intracellularly to block the conversion of corticosterone to aldosterone. Taken together, these results indicate that, in amphibians extracellular calcium is essential for the action of ACTH, either for the binding of the hormone to its receptor and/or for the transduction of the information from hormone-receptor complex to the adenylate cyclase moiety and that the mechanism of action of angiotensin does not involve calcium uptake by adrenocortical cells.

Adrenal Glands↗
PubMed1986

High yield photoaffinity labeling of angiotensin II receptors.

The angiotensin II analogues, [Sar1, (4'-N3)Phe8]All and [Sar1, (4'-N3)D-Phe8]All were synthetized and prepared in their iodinated and radioiodinated forms. On rat liver membranes 125I-SarN3PheAll and 125I-SarN3DPheAll labeled a single population of sites (maximal binding capacity, 1.13 +/- 0.16 pmol/mg of protein) with high affinity (dissociation constants of 0.18 +/- 0.05 and 0.37 +/- 0.16 nM, respectively) and high specificity (nonspecific binding less than 10% of total binding for an 0.8 fractional receptor occupancy). 125I-SarN3PheAll and 125I-SarN3DPheAll allowed photoaffinity labeling of liver angiotensin receptors with high efficiency. The yield of photoaffinity labeling was independent of fractional receptor occupancy. 125I-SarN3PheAll and 125I-SarN3DPHeAll allow recovery in a soluble and covalently labeled form of about 40% of the total number of angiotensin receptors from rat liver membranes. For this reason they can be considered as potentially very useful tools for the purification of angiotensin receptors. The material covalently labeled with the antagonist 125I-SarN3DPHeAll was eluted from an Ultrogel ACA34 column as a homogeneous peak (Stoke's radius: 5.5 +/- 0.2 nm). The material covalently labeled with the agonist 125I-SarN3PHeAll was more heterogeneous. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of 125I-SarN3PheAll- and 125I-SarN3DPHeAll-labeled material revealed a single component of 63,000 molecular weight.

Adenylyl Cyclases↗