Biomedical subjects

D Hoyer

Publications and source records attributed to D Hoyer.

At least 19 recordsLinked to original sources

PubMed2000

Design and synthesis of potent thiol-based inhibitors of endothelin converting enzyme-1.

Through directed screening of compounds prepared as metalloprotease inhibitors a compound, CGS 30084, that had potent endothelin converting enzyme-1 (ECE-1) in vitro inhibitory activity (IC50 = 77 nM) was identified. Herein we report the synthesis and optimization of ECE-1 inhibitory activity of additional analogues from this lead. Compound 3c, the thioacetate methyl ester derivative of compound 4c, was found to be a long acting inhibitor of ECE-1 activity in rats after oral administration.

Animals↗

PubMed2000

Cloning, expression and pharmacological characterisation of the mouse somatostatin sst(5) receptor.

The mouse somatostatin (somatotropin release inhibiting factor, SRIF) sst(5) receptor coding sequence was cloned from a mouse BALB/c genomic library. It shows 97% and 81% homology with the corresponding rat and human receptors, respectively. The msst(5) receptor messenger RNA (mRNA) is present at low levels in the adult mouse brain, with significant expression in a few nuclei only, e.g. in the septum (lateral septal nuclei) or the amygdala (medial amygdaloid nucleus); very few signals were observed in the mesencephalon, metencephalon, and myelencephalon (except the dorsal motor nucleus of the vagus nerve). The msst(5) receptor was stably expressed in the hamster fibroblast cell line CCL39-SRE-Luci, which harbours the luciferase reporter gene driven by the serum responsive element. [(125)I]LTT-SRIF-28 ([Leu(8), D-Trp(22), (125)I-Tyr(25)]-SRIF-28), [(125)I]Tyr(10)-CST, [(125)I]CGP 23996, and [(125)I]Tyr(3)-octreotide labelled msst(5) receptors with high affinity (pK(d) values: 11.0, 10.15, 9.75 and 9.43) and in a saturable manner, but defined different Bmax values: 697, 495, 540 and 144 fmoles/mg, respectively. [(125)I]LTT-SRIF-28-labelled sites displayed the following rank order: SRIF-28> rCST-14> somatuline > CGP-23996= SRIF-14= octreotide, whereas [(125)I]Tyr(3)-octreotide-labelled sites displayed a different profile: octreotide > SRIF-28> rCST-14= somatuline > SRIF-14> CGP-23996. The pharmacological profiles determined with [(125)I]LTT-SRIF-28, [(125)I]CGP 23996 and [(125)I]Tyr(10)-CST correlated highly significantly (r(2) =0.88-0.99), whereas [(125)I]Tyr(3)-octreotide binding was rather divergent (r(2) =0.77). Also, human and mouse sst(5) receptor profiles are very different, e. g. r(2) =0.385 for [(125)I]Tyr(10)-CST and r(2) =0.323 for [(125)I]LTT-SRIF-28-labelled sites. Somatostatin induces expression of luciferase reporter gene in CCL39-SRE-Luci cells. The profile was consistent with a msst(5) receptor-mediated effect although apparent potency in the luciferase assay was much reduced compared to radioligand binding data: Octreotide = SRIF-28> rCST-14= SRIF-14= CGP-23996. Octreotide, SRIF-28, BIM23052 and D Tyr Cyanamid 154806 behaved as full or nearly full agonists in comparison to SRIF-14, whereas the other compounds had relative efficacies of 40 to 70%. The present study shows that agonists radioligands define apparently different receptor populations in terms of number of sites and pharmacological profile in cells expressing a single recombinant receptor. These variations suggest that the conformation of the ligand receptor complex may vary depending on the agonist. Further, the msst(5) receptor, although primarily coupled to Gi/Go proteins, is able to stimulate luciferase gene expression driven by the serum responsive element. Finally, it is suggested that putative sst(2) selective agonists e.g. octreotide, RC160 or BIM23027 show similar or higher potency at msst(5) receptors than SRIF-14.

Amino Acid Sequence↗

PubMed2000

Potent and selective non-peptidic inhibitors of endothelin-converting enzyme-1 with sustained duration of action.

Potent and selective non-peptidic inhibitors of human endothelin-converting enzyme-1 (ECE-1) have been designed as potential modulators of endothelin (ET-1) production in vivo. Because of its unique structural characteristics and long duration of action in vivo, the dual ECE-1 and neutral endopeptidase 24.11 (NEP) inhibitor, CGS 26303, was selected as an attractive lead for further optimization of potency and selectivity. Replacement of the P(1)' biphenyl substituent of CGS 26303 by a conformationally restricted 3-dibenzofuranyl group led to more potent and more selective ECE-1 inhibitors, such as the tetrazole 27. The remarkable effect of this P(1)' modification allowed for the first time phosphonomethylcarboxylic acids, such as 29, to display both potent (IC(50) = 22 nM) and selective (104-fold vs NEP) ECE-1 inhibition. Chemoenzymatic syntheses of the new alpha-amino acid (S)-3-dibenzofuran-3-ylalanine intermediate were developed, and improved procedures to generate substituted alpha-aminoalkylphosphonic acids were devised to support the production of various analogues. Although additional gains in intrinsic ECE-1 inhibitory potency could occasionally be achieved by addition of a P(1) side chain, these compounds (e.g. 43a) showed poor functional activity in vivo in the big ET-1 pressor test. Phosphonoalkyl dipeptides featuring 3-dibenzofuranyl groups in both the P(1)' and P(2)' positions were also very potent ECE-1 inhibitors, albeit lacking the desired selectivity against NEP. Functionally, 27and 29 were the two most efficacious compounds from this study, producing sustained inhibition of ECE-1 activity in rats, as measured by their ability to block the hypertensive effects induced by big ET-1. This profile was similar to that of a potent ET(A)/ET(B) dual receptor antagonist, SB 209670. Due to their favorable in vitro and in vivo profiles, 27 (CGS 34043) and 29 (CGS 35066) constitute new pharmacological tools useful in assessing the role of ECE-1 in pathological conditions.

Animals↗

PubMed2000

Syntheses and biological activities of sandostatin analogs containing stereochemical changes in positions 6 or 8.

In a continuation of our research efforts on the design and synthesis of novel peptidomimetic structures, we have synthesized a series of sandostatin amide analogs in which stereoisomers of threonine and beta-hydroxyvaline(beta-Hyv) are employed. The analogs D-Phe1-c[Cys2-Phe3-D-Trp4-Lys5-Xaa6-Cys 7]-Xbb8-NH2 (Xaa = allo-Thr, D-allo-Thr, D-beta-Hyv, beta-Hyv, D-Thr, and Xbb = Thr or Xaa = Thr and Xbb = allo-Thr, D-allo-Thr, beta-Hyv, D-Thr) explore the effects on biological activity of stereochemical modifications and beta-methylation at positions 6 or 8. By these modifications, we examine the role of the two residues in binding to somatostatin receptors. We describe the synthesis and biological activity of these analogs. In combination with the results of the conformational analysis, this study provides new insights into the structural requirements for the binding affinity of somatostatin amide analogs to somatostatin receptors [Mattern et al., Conformational analyses of sandostatin analogs containing stereochemical changes in positions 6 or 8].

Animals↗

PubMed2000

Cloning, expression, functional coupling and pharmacological characterization of the rat dopamine D4 receptor.

It has been difficult to observe functional coupling of the D4 receptor to second messenger systems and a robust functional assay system for this receptor is still lacking. In the present study, the rat dopamine D4 receptor was cloned from rat retina. Sequence comparison revealed identity with the published sequence of Ashgari and co-workers, including the two amino acid insertions (V-Q) at position 92 which are not present in the published sequence of O'Malley and coworkers. The rat dopamine D4 receptor was stably expressed in Chinese hamster lung fibroblast CCL39 cells. [3H]spiperone saturation binding yielded a Bmax of 2,370+/-546 fmol/mg protein and a pKD of 8.74+/-0.14 (n=4). Forskolin-stimulated cAMP accumulation was inhibited by dopamine (Emax 61+/-1% inhibition of forskolin-stimulated levels, pEC50 7.33+/-0.06, n=23). A similar concentration-dependent inhibition was observed with the dopamine D2-like receptor agonists quinpirole and 7-OH-DPAT which elicited nearly the same Emax as dopamine. By contrast, apomorphine and a number of compounds with reported affinity for human dopamine D4 receptors (PD168077, U-101958, SDZ GLC 756, L-745,870 and NGD 94-1) behaved as partial agonists (Emax ranging between 26% and 56% of that of dopamine). The agonist effect of dopamine was completely blocked by preincubation with pertussis toxin, no further accumulation of cAMP above the forskolin-stimulated levels being observed. Antagonist pKB-values obtained against dopamine in this system were: 8.55+/-0.19 (n=3) for the partial agonist L-745,870, 8.38+/-0.23 (n=5) for spiperone, 7.18+/-0.17 (n=4) for haloperidol, 7.04+/-0.13 (n=4) for clozapine and <6 for raclopride. Other functional assays applicable were stimulation of [35S]GTPgammaS binding, extracellular acidification rate and a serum-responsive element using luciferase expression as a reporter gene. However, the receptor did not couple to phosphatidylinositol turnover or to intracellular Ca2+. Thus, expression of the rat dopamine D4 receptor in CCL39 cells provided several functional assay systems, of which inhibition of cAMP appeared to be the most robust one. These functional models can be used to evaluate the activity of compounds at the rat dopamine D4 receptor.

Animals↗

PubMed2000

[Cardiorespiratory desynchronization after acute myocardial infarct].

The prognosis of cardiac diseases can be estimated from the variability of regulation parameters of the cardiovascular system. Changes in the variability of a regulation parameter causes disturbances in the synchronisation of interacting control loops. Conclusions about the severity of the underlying functional impairment can be drawn from these disturbances. This study investigates the synchronisation of the control loops of the heart rate and respiration (cardiorespiratory synchronisation, CRS) after acute myocardial infarction. We investigated 43 patients after myocardial infarction and 27 healthy controls. To quantify the CRS the synchronisation in phase of respiration and heart rate was assessed. The heart rate variability (HRV) was also assessed. Patients after myocardial infarction have a significantly reduced HRV and CRS. There is a non-linear relationship between HRV and CRS. Patients with left ventricular enlargement and reduced left ventricular ejection fraction (< or = 45%) significantly differed from the other infarct patients and controls in CRS but not in HRV. They had a marked degree of cardiorespiratory desynchronisation and were identified by a threshold value. CRS is a measure of the interaction of respiration control and heart rate control. After myocardial infarction, a reduction of the HRV can be observed. The desynchronisation of the control loops of respiration and heart rate especially appears in large infarcts. This can be quantitatively assessed by the method presented.

Adult↗

PubMed2000

Recovery of emotional behaviour in neural cell adhesion molecule (NCAM) null mutant mice through transgenic expression of NCAM180.

In the present study we further investigate functions of the neural cell adhesion molecule (NCAM) in the mature central nervous system and its implications for animal behaviour. To this end we generated transgenic mice expressing the major NCAM isoform with the largest cytoplasmic domain, NCAM180, under control of a promoter for the small form neurofilament gene. Transgenic mice were also bred with mice deficient in endogenous NCAM (Ncam-/- mice) so that effects of NCAM180 could be analysed in the presence and absence of endogenous NCAM. While overexpression of transgenic NCAM180 was without apparent behavioural or morphological effect, its expression in Ncam-/- mice counteracted NCAM ablation-induced aggressive, anxiety-like and antidepressant-like behaviour. It furthermore prevented a hypersensitivity of Ncam-/- mice to the anxiolytic serotonin1A (5-HT1A) receptor agonist buspirone. Such recovery of emotional behaviour and behavioural 5-HT1A response occurred in spite of misdevelopment of the olfactory bulb and hippocampus that is characteristic of Ncam-/- mice, and without an apparent change in the expression of 5-HT1A binding sites in the brain. Hippocampus- and amygdala-dependent learning, though disturbed in Ncam-/- mice, remained unaffected by the transgenic NCAM180. We suggest an involvement of NCAM180-mediated cell recognition processes in the serotonergic modulation of emotional behaviour in adult mice.

Aggression↗

PubMed2000

Effect of mild hypothermia on cerebral oxygen uptake during gradual cerebral perfusion pressure decrease in piglets.

OBJECTIVE: To study the effect of mild hypothermia on cerebral oxygen metabolism and brain function in piglets during reduced cerebral blood flow because of gradual reduction of the effective cerebral perfusion pressure (CPP). DESIGN: Comparison of two randomized treatment groups: normothermic group (NT; n = 7) and hypothermic group (HT; n = 7). SETTING: Work was conducted in the research laboratory of the Institute for Pathophysiology, Friedrich Schiller University, Jena, Germany. SUBJECTS: Fourteen piglets (14 days old) of mixed German domestic breed. INTERVENTION: Animals were anesthetized and mechanically ventilated. An epidural balloon was gradually inflated to increase intracranial pressure to 25 mm Hg, 35 mm Hg, and 45 mm Hg every 30 mins at adjusted mean arterial blood pressures. After determination of baseline CPP (NT, 79+/-14 mm Hg; HT, 84+/-9 mm Hg), CPP was reduced to approximately 70%, 50%, and 30% of baseline (NT, 38.1+/-0.5 degrees C; HT, 31.7+/-0.5 degrees C). MEASUREMENTS AND MAIN RESULTS: Every 25 mins after the gradual CPP reductions. Mild hypothermia induced a reduction of the cerebral metabolic rate of oxygen (CMRO2) to 50%+/-15% of baseline values (baseline values, 352+/-99 micromol x 100 g(-1) x min(-1)) (p < .05). Moreover, the electrocorticogram was altered to a pattern of reduced delta activity (p < .05) but unchanged higher frequency activity. The cerebral oxygen balance in HT animals remained improved until CPP reduction to 50%, indicated by a reduced cerebral arteriovenous difference of oxygen but elevated brain tissue Po2 (p < .05). Further CPP reduction gave rise to a strong CMRO2 reduction (NT, 19+/-21%; HT, 15+/-15%; p < .05). However, the high-frequency band of electrocorticogram was less reduced in hypothermic animals (p < .05). CONCLUSIONS: Mild whole body hypothermia improves cerebral oxygen balance by reduction of brain energy demand in juvenile piglets. The improvement of brain oxygen availability continues during a mild to moderate CPP decrease. A loss of the difference in CMRO2 between the hypothermic and normothermic piglets together with the fact that brain electrical activity was less suppressed under hypothermia during severe cerebral blood flow reduction indicates that hypothermic protection may involve some other mechanisms than reduction of brain oxidative metabolism.

Analysis of Variance↗

PubMed2000

A new approach to uncover dynamic phase coordination and synchronization.

Phase attractive maps are an essential mechanism of multi-stable systems such as found in coupled neuronal oscillators. An essential feature of this type of dynamic nonlinear coordination is dynamic phase synchronization. The identification of dynamic phase synchronizations is complicated due to changing frequency ratios of synchronized intervals, other nonstationarities, and noise. In order to overcome these problems the momentary phase relations and their statistics were analyzed by several authors. In this way phase synchronizations also in chaotic and noisy oscillating systems could be uncovered. We propose a novel method which avoids one essential limitation of these approaches, namely the necessity of presetting particular frequency ratios of interest. The proposed novel method was validated by its application to a simulated driven neuronal generator during the transition period between different synchronization modes and to dynamically coupled components of sympathetic nerve discharges.

Action Potentials↗

PubMed2000

Involvement of the Sst1 somatostatin receptor subtype in the intrahypothalamic neuronal network regulating growth hormone secretion: an in vitro and in vivo antisense study.

Five somatostatin (SRIH) receptors (sst1-5) have been cloned. Recent anatomical evidence suggests that sst1 and sst2 may be involved in the central regulation of GH secretion. Given the lack of specific receptor antagonists, we used selective antisense oligodeoxynucleotides (ODNs) to test the hypothesis that one or both of these subtypes are involved in the intrahypothalamic network regulating pulsatile GH secretion. In mouse neuronal hypothalamic cultures the proportion of GHRH neurons coexpressing sst1 or sst2 messenger RNAs (mRNAs) was identical. In contrast, sst1 mRNAs were more often present than sst2 in SRIH-expressing neurons. Firstly, sst1 antisense ODN in vitro treatment abolished sst1, but not sst2, receptor modulation of glutamate sensitivity and decreased sst1, but not sst2, mRNAs. The reverse was true after treatment with sst2 antisense. Sense ODNs did not alter the effects of SRIH agonists. In a second series of experiments, nonanaesthetized adult male rats were infused for 120 h intracerebroventricularly with ODNs. Only the sst1 antisense ODN diminished the amplitude of ultradian GH pulses without modifying their frequency. In parallel, sst1 antisense ODN strongly diminished sst1 immunoreactivity in the anterior periventricular nucleus and median eminence, as well as sstl periventricular nucleus mRNA levels. The effectiveness of the sst2 antisense ODN was attested by the inhibition of hypothalamic binding of [125I]Tyr0-D-Trp8-SRIH. Scrambled ODNs had no effect on GH secretion or on sst mRNAs or SRIH binding levels. These results favor a preferential involvement of sst1 receptors in the intrahypothalamic regulation of GH secretion by SRIH.

Animals↗

PubMed2000

Somatostatin receptors and the potential use of Sandostatin to interfere with vascular remodelling.

Graft vessel disease (GVD) is a major cause of graft loss after the first year following transplantation. GVD is a complex, multifunctional process that involves immunological as well as non-immunological events such as ischaemia/reperfusion injury. An important target cell to interfere with the development of GVD is the smooth muscle cell (SMC). Somatostatin (SRIF) analogues have been shown previously to inhibit the proliferation of SMC in vitro and in vivo. We provide evidence that Sandostatin, an octapeptide SRIF analogue that is known to have anti-proliferative properties on SMC proliferation, inhibits vascular remodelling in a rat angioplasty model. Furthermore, in two allotransplantation models, Sandostatin effectively interferes with the development of signs of chronic rejection/GVD. The role of the different SRIF receptor subtypes in chronic graft rejection is currently under investigation.

Animals↗

PubMed1999

Anxiety and increased 5-HT1A receptor response in NCAM null mutant mice.

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety-like behavior of homozygous (NCAM-/-) and heterozygous (NCAM/-) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety-like behavior was reduced in both NCAM+/+ and NCAM-/- mice by systemic administration of the benzodiazepine agonist diazepam and the 5-HT1A receptor agonists buspirone and 8-OH-DPAT. However, NCAM-/- mice showed anxiolytic-like effects at lower doses of buspirone and 8-OH-DPAT than NCAM+/+ mice. Such increased response to 5-HT1A receptor stimulation suggests a functional change in the serotonergic system of NCAM-/- mice, likely involved in the control of anxiety and aggression. However, 5-HT1A receptor binding and tissue content of serotonin and its metabolite 5-hydroxyindolacetic acid were found unaltered in every brain area of NCAM-/- mice investigated, indicating that expression of 5-HT1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM-/- mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5-HT1A receptors and inwardly rectifying K+ channels as the respective effector systems.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1999

Structural and compositional determinants of cortistatin activity.

Cortistatin-14 (CST-14) is a putative novel neuropeptide that shares 11 of its 14 residues with somatostatin-14 (SRIF-14), yet its effects on sleep physiology, locomotor behavior and hippocampal function are different from those of somatostatin. We studied the structural basis for cortistatin's distinct biological activities. As with SRIF-14, CST-14 does not show any preferred conformation in solution, as determined by circular dichroism and nuclear magnetic resonance. Synthetic cortistatin analogs were designed and synthesized based on the cyclic structure of octreotide. Biological assays were carried out to determine their binding affinities to five somatostatin receptors (sstl-5) and their ability to produce changes in locomotor activity and to modulate hippocampal physiology and sleep. The results show that the compound with N-terminal proline and C-terminal lysine amide exhibits cortistatin-like biological activities, including reduction of population spike amplitudes in the hippocampal CA1 region, decrease in locomotor activity and enhancement of slow-wave sleep 2. These findings suggest that both proline and lysine are necessary for cortistatin binding to its specific receptor.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and pharmacological characterization of a somatostatin receptor subtype in the gymnotiform fish Apteronotus albifrons.

The actions of the various forms of somatostatin (SRIF), including those of the tetradecapeptide SRIF(14), are mediated by specific receptors. In mammals, five subtypes of SRIF receptors, termed sst(1-5), have been cloned. Using a combination of reverse transcriptase-polymerase chain reaction and genomic library screening in the gymnotiform fish Apteronotus albifrons, a gene encoding the first-known nonmammalian SRIF receptor has been isolated. The deduced amino acid sequence displays 59% identity with the human sst(3) receptor protein; hence, the gene is termed "Apteronotus sst(3)." The predicted protein consists of 494 amino acid residues exhibiting a putative seven-transmembrane domain topology typical of G protein-coupled receptors. A signal corresponding to the Apteronotus sst(3) receptor was detected in brain after amplification of poly(A)(+)-RNA by reverse transcriptase-polymerase chain reaction, but not by Northern blot analysis or in situ hybridization, suggesting a low level of expression. Membranes prepared from CCL39 cells stably expressing the Apteronotus sst(3) receptor gene bound [(125)I][Leu(8),d-Trp(22), (125) I-Tyr(25)]SRIF(28) with high affinity and in a saturable manner (B(max) = 4470 fmol/mg protein; pK(D) = 10.5). SRIF(14) and various synthetic SRIF receptor agonists produced a dose-dependent inhibition of radioligand binding, with the following rank order of potency: SRIF(14) approximately SRIF(28) > BIM 23052 > octreotide > BIM 23056. Under low stringency conditions, an Apteronotus sst(3) probe hybridized to multiple DNA fragments in HindIII or EcoRI digests of A. albifrons DNA, indicating that the Apteronotus sst(3) receptor is a member of a larger family of Apteronotus SRIF receptors.

Amino Acid Sequence↗

PubMed1999

Pharmacological characterisation of 5-HT receptors positively coupled to adenylyl cyclase in the rat hippocampus.

The pharmacological properties of 5-hydroxytryptamine (5-HT) receptors positively coupled to adenylyl cyclase in the rat hippocampus were investigated using selective agonists and antagonists. 5-HT (0.008-125 microM) stimulated cyclic AMP formation in homogenates of rat hippocampus in a concentration-dependent manner. The maximal increase in cyclic AMP formation occurred at 1 microM (141+/-6%) and the half-maximal effect (EC50) at 50+/-22 nM. Cyclic AMP accumulation induced by 1 microM 5-HT was partly inhibited by the selective 5-HT1A receptor antagonist WAY 100,635 (1 microM), the selective 5-HT4 receptor antagonist SB 203,186 (1 microM), and the 5-HT2A/c/ 5-HT7 receptor antagonist mesulergine (25 microM). WAY 100,635, SB 203,186 and mesulergine inhibited the effect of 5-HT (1 microM) by 47%, 33% and 49%, respectively. The combination of WAY 100,635 (1 microM) with SB 203,186 (1 microM) or mesulergine (25 microM) resulted in stronger inhibition than with each antagonist alone, and the combination of all three antagonists produced almost total blockade (95%) of 5-HT-induced cyclic AMP accumulation. 5-Carboxamidotryptamine (5-CT; 0.008-125 microM), a 5-HT1/5-HT7 receptor agonist, and SDZ 216-454 (0.008-125 microM), a selective 5-HT4 receptor agonist, concentration-dependently stimulated cyclic AMP formation, but the maximal effect of each agonist was smaller than that of 5-HT alone. SDZ 216-454 (5 microM) and 5-CT (5 microM) in combination stimulated cyclic AMP formation in an additive manner. 8-OH-PIPAT and 8-OH-DPAT, two selective 5-HT1A agonists, produced a small but significant increase in cyclic AMP formation at concentrations above 0.04 microM and 10 microM, respectively. These findings suggest that at least three 5-HT receptor subtypes, i.e. 5-HT1A, 5-HT7 and 5-HT4 receptors, are involved in mediating 5-HT-induced cyclic AMP formation in rat hippocampus.

Adenylyl Cyclases↗

PubMed1999

Characterisation of human recombinant somatostatin receptors. 1. Radioligand binding studies.

Human somatostatin receptor subtypes 1-5 (sst1-5) were characterised using the agonist radioligands [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996 and [125I][Tyr3]octreotide in stably transfected Chinese hamster lung fibroblast cells (CCL39 cells). The radioligands used labelled saturable and high-affinity populations of sites in each instance; at sst1-4 receptors maximum binding (Bmax) was roughly equivalent. By contrast, at sst5 receptors Bmax determined with [125I]CGP 23996 and [125I][Tyr3]octreotide was significantly lower (two-and eightfold) compared with [125I]LTT-SRIF28 and [125I][Tyr10]CST14. Experiments were performed with the stable GTP-analogue guanylylimidodiphosphate (GppNHp) to establish guanine nucleotide sensitivity of agonist binding to sst1-5 receptors. The sensitivity towards GppNHp was quite variable depending on receptor and/or ligand. At sst1 and sst4 receptors, GppNHp produced little effect overall, whereas binding to sst3 and sst2 receptors was reduced by 70 and >80%, respectively. At sst5 receptors, the binding of [125I]LTT-SRIF28 and [125I][Tyr10]CST14 was only slightly affected by GppNHp, while [125I]CGP 23996 and [125I][Tyr3]octreotide binding was almost entirely inhibited. Thus, [125I][Tyr3]octreotide labelled about 26-fold less sst5 receptors than [125I]LTT-SRIF28, in the presence of 10 microM GppNHp. These discrepancies in guanine nucleotide sensitivity, were confirmed in GppNHp competition experiments. Competition studies were performed at the five receptors labelled with the different radioligands to establish their respective pharmacological profiles: the rank order of affinity was largely radioligand-independent at sst1-4 receptors, in contrast to sst5 receptors where it was radioligand-dependent. Thus, the pharmacological profile of [125I]LTT-SRIF28- and [125I][Tyr10]CST14-labelled sst5 sites correlated highly significantly, but did not correlate with the affinity profiles defined with [125I]CGP 23996 and [125I][Tyr3]octreotide binding to sst5 receptors. Depending on the agonist radioligand used and the receptor studied, it would appear that binding can be essentially to a guanine nucleotide-sensitive state (e.g. sst2 or sst3), a guanine nucleotide-insensitive state (sst1 or sst4) or a mixture of both (sst5); in the latter case, each radioligand defining a more or less different rank order of affinity at the same receptor. In summary, the differences in agonist receptor binding and guanine nucleotide sensitivity cannot be explained by the ternary complex model or its variations, but rather suggest the existence of multiple agonist-specific receptor states which vary from one receptor to another.

Animals↗

PubMed1999

Characterisation of human recombinant somatostatin receptors. 2. Modulation of GTPgammaS binding.

G protein activation by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and analogues of these neuropeptides was investigated at human somatostatin receptor subtypes 1-5 (sst1-5) stably expressed in CCL39 Chinese hamster lung fibroblast cells by measuring agonist-stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding. [35S]GTPgammaS binding was assessed in the presence of 100 mM NaCl and 1 microM GDP, although higher Emax and/or pEC50 values may have been obtained under other conditions, but at the expense of lower absolute stimulation or signal/noise ratio. SRIF14 stimulated [35S]GTPgammaS binding to 162, 220, 148 and 266% of control levels via sst2, sst3, sst4 and sst5 receptors, respectively. At sst1 receptors, SRIF14 produced only a limited stimulation (Emax 115%). Hence sst1 receptors were not subjected to further [35S]GTPgammaS binding experiments. [35S]GTPgammaS binding assays were then performed with sst2-5 receptors. Most of the peptide analogues stimulated [35S]GTPgammaS binding in sst2-5 receptor-expressing cells. BIM 23056 behaved as an antagonist on SRIF14-induced [35S]GTPgammaS binding with an apparent pKBs of 6.33 and 5.84 at hsst3 and hsst5 receptors respectively, whereas neither agonism nor antagonism could be shown (at 1 microM) at sst2 or sst4 receptors. The effect at sst5 receptors was not surmountable and needs further investigations. The so-called "antagonist" SA, was devoid of antagonist activity at sst2 or sst3 receptors, whereas it was almost a full agonist at sst4 and sst5 receptor-mediated [35S]GTPgammaS binding. The [35S]GTPgammaS-binding profiles of hsst2-5 receptors were compared with their respective radioligand binding profiles. For sst4 and sst5 receptors, the rank order of affinity of all tested radioligands correlated highly significantly with [35S]GTPgammaS binding (r = 0.814-0.897). At sst3 receptors, [35S]GTPgammaS binding correlated somewhat less with binding profiles obtained with [125I][Tyr10]CST14 and [125I]CGP 23996 than with [125I]LTT-SRIF28 (r = 0.743, 0.757 and 0.882, respectively). At sst2 receptors, [35S]GTPgammaS binding correlated with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I][Tyr3]octreotide binding profiles (r = 0.596-0.699), but not with [125I][Tyr10]CST14 binding. The present [35S]GTPgammaS binding data combined to previous radioligand binding results obtained in cells expressing human SRIF receptors, suggest that at any given receptor, agonists' rank orders of potency (not to mention absolute affinity values which vary profoundly) are not as strictly ordered as may be anticipated. We are investigating these aspects further by analysing additional signalling pathways.

Animals↗

PubMed1999

Characterisation of human recombinant somatostatin receptors. 3. Modulation of adenylate cyclase activity.

The five human somatostatin receptor subtypes (hsst1-5) were stably expressed in CCL39 cells (Chinese hamster lung fibroblast cells) to study the inhibition of forskolin-stimulated adenylate cyclase (FSAC) activity induced by somatostatin (somatotropin release inhibiting factor, SRIF), cortistatin (CST) and SRIF peptide analogues. Inhibition of FSAC was observed with all five receptors, although the maximal effects produced by SRIF14 varied from around 40% (sst1, sst2, sst4) to 67% (sst3, sst5) reflecting to some extent differences in receptor density (Bmax values published in accompanying paper, this journal). SRIF28 was slightly more potent than SRIF14 to inhibit FSAC at all five receptors, although the potency of the natural peptides SRIF14, SRIF28 and CST17 was generally similar with pEC50-values ranging from 7.5 to 8.7 depending on receptor and peptide. At SRIF1 receptors (sst2, sst3, sst5) most of the peptide analogues displayed full agonism (with some exceptions e.g. BIM 23056 at sst1-3 and sst5 receptors, and L362,855 and cycloantagonist SA at sst3 receptors), whereas at SRIF2 receptors these analogues tended to behave as partial agonists. BIM 23056 was an antagonist at sst3 receptors (antagonist binding constant pKB = 6.33), but not at other receptors. The AC inhibition profiles of sst1-5 receptors were compared with the different radioligand binding profiles as well as with [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTPgammaS) binding profile for sst2-5 receptors. High correlations were observed between FSAC inhibition, radioligand binding and [35S]GTPgammaS binding profiles at sst3, sst4 and sst5 receptors; by contrast, correlation coefficients at sst1 and sst2 receptors were low, and the binding profiles of [125I][Tyr10]CST14 correlated poorly. In line with these findings, the FSAC inhibition and [35S]GTPgammaS binding correlated poorly at sst2 receptors (sst1 receptors show no significant induction of [35S]GTPgammaS binding). The apparent lack of, or only weak, relationship between FSAC, radioligand or [35S]GTPgammaS binding observed for some SRIF receptors suggests that different active states may exist for these receptors, which may favour one transduction cascade over others.

Adenylyl Cyclase Inhibitors↗