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D Hoyer

Publications and source records attributed to D Hoyer.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1999

Characterisation of human recombinant somatostatin receptors. 4. Modulation of phospholipase C activity.

Total [3H]phosphoinositide (IPx) accumulation, a measure of phospholipase C (PLC) activity, induced by somatostatin (somatotropin release-inhibiting factor, SRIF) and cortistatin (CST) analogues was studied at human somatostatin receptor subtypes 1-5 (hsst1-5) recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. SRIF14 (10 microM) stimulated total [3H]-IPx production 200% and 1070% over basal levels, and increased intracellular Ca2+ ([Ca2+]i) 1600% and 2790%, in cells expressing hsst3 and hsst5 receptors, respectively. The SRIF14-stimulated IPx production was partly blocked by 100 ng/ml pertussis toxin (PTX) (30% and 15% inhibition, respectively). At hsst1, hsst2, and hsst4 receptors, only weak or no stimulation of PLC activity was found (Emax = 114%, 122%, and 102%, respectively). Consequently, hsst3 and hsst5 receptors were subjected to more detailed studies to establish pharmacological profiles of PLC stimulation. At hsst3 receptors, the relative efficacies of most ligands were in the same range (maximum response Emax = 218-267%). At hsst5 receptors Emax varied over a broad range, seglitide, CST17, SRIF28 displaying almost full agonism compared to SRIF14, whereas octreotide and BIM 23052 showed very low partial agonism. BIM 23056 behaved as an antagonist on SRIF14-induced total [3H]-IPx accumulation with a pKB (negative logarithm of antagonist binding constant) of 6.74 at hsst3 receptors, and of 6.94 at hsst5 receptors. The putative cycloantagonist SA showed weak antagonist activity on SRIF14-induced total [3H]-IPx levels at hsst3 (pKB = 5.85), but not at hsst5 receptors. The [3H]-IPx accumulation profiles at sst3/sst5 receptors were compared to their respective radioligand binding ([125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide binding), to [35S]GTPgammaS binding, and to forskolin-stimulated adenylate cyclase (FSAC) inhibition profiles determined previously in CCL39 cells. The different affinity profiles correlated relatively well at both receptor subtypes with PLC activation (sst3: r = 0.90-0.97; sst5: r = 0.80-0.87). However, [35S]GTPgammaS binding correlated only minimally with stimulation of [3H]-IPx levels at sst5 receptors (r = 0.59), but rather well at sst3 receptors (r = 0.80). A moderate correlation was also observed between inhibition of FSAC activity and stimulation of PLC activity for hsst3 and hsst5 receptors with correlation coefficients of 0.85 and 0.70, respectively. In summary, most SRIF analogues behave as full agonists at hsst3 receptors and agonist-induced phosphoinositide turnover correlates well with radioligand binding, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, all measured in CCL39 cells. By contrast, at hsst5 receptors, most SRIF analogues behave as intermediate or very low partial agonists (although receptor levels are comparatively high, 7000 vs. 400 fmol/mg), and the agonist-induced phosphoinositide turnover correlates rather poorly with radioligand binding, [35S]GTPgammaS binding or inhibition of adenylate cyclase activity, all measured in the same cell line. Agonist-induced phosphoinositide turnover, [35S]GTPgammaS binding and inhibition of adenylate cyclase activity, show differences both in the rank orders of potency and relative efficacy at hsst3 and markedly at hsst5 receptors, suggesting either that PLC activity is functionally irrelevant or, more probably, that agonist-dependent receptor trafficking is taking place in CCL39 cells.

Adenylate Cyclase Toxin↗
PubMed1999

Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family: atypical pharmacological features.

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst3 receptors; [125I]LTT-SRIF28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF28), [125I]Tyr10-cortistatin, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled the fsst3 receptor with high affinity (pKd values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different Bmax values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst3 receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide > or = BIM 23052 > or = L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I]Tyr10-cortistatin correlated highly significantly (r = 0.96-0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r = 0.78-0.81). Further, [125I]Tyr3-octreotide- and [125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [125I]LTT-SRIF28 and [125I]Tyr10-cortistatin-labelled sites, although there were exceptions. [125I]LTT-SRIF28-binding to fsst3 receptors and human sst1-5 receptors was compared; the fsst3 binding profile correlated better with the hsst5- than with the hsst3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst3 receptor to Gialpha and/or Goalpha. [125I]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [125I]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst3 receptor, in agreement with expression of the fsst3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst3 receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst3 is closer to that of hsst5 than hsst3 receptors, as may be found when comparing very distantly related species. The fsst3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.

Adenylyl Cyclases↗
PubMed1999

Hypothermia related changes in electrocortical activity at stepwise increase of intracranial pressure in piglets.

Recent experimental studies have demonstrated that mild hypothermia can be effective in the control of intracranial hypertension. However, investigations to analyze the effects of hypothermia on changes in brain oxygen metabolism and electrocortical activity caused by increased intracranial pressure (ICP) are lacking. We examined the effects of mild hypothermia on electrocorticogram (ECoG) in combination with measurement of regional cerebral blood flow (CBF) and estimation of brain oxygen metabolism during stepwise increase of ICP. For this purpose thirteen female piglets (14 days old, 4-5 kg b.w.) were anaesthetized and mechanically ventilated. An epidural balloon was gradually inflated in order to increase ICP to 25 mmHg, 35 mmHg and 45 mmHg every 30 minutes at adjusted mean arterial blood pressures (MAP). This procedure resulted in gradual cerebral perfusion pressure (CPP) reduction of about 70%, 50%, and 30% of baseline [baseline CPP: normothermia (NT) 80+/-3 mmHg; hypothermia (HT) 84+/-3 mmHg]. Control animals were maintained in a normothermic state (38.6+/-0.2 degrees C). HT animals were surface cooled and maintained at 31.9+/-0.1 degrees C. ECoG, regional CBF, cerebral oxygen delivery (cDO2) and the cerebral metabolic rate of oxygen (CMRO2) were estimated during the normothermic period, after hypothermic stabilization, and after the gradual CPP reductions. The baseline ECoG showed the typical delta-dominated frequency pattern for isoflurane anaesthesia. At the hypothermic level, a frequency shift was seen from delta activity towards the higher frequencies (theta- and alpha activity) and the total spectral power was significantly reduced (56+/-17% from baseline, p < 0.05). the cortical CBF decreased markedly to 67+/-10% (p < 0.05), whereas the medulla oblongata blood flow increased slightly. During controlled increase of ICP by regional mass expansion from epidural balloon inflation, we found at mild and moderate stages of ICP increase (25 and 35 mmHg) only minimal changes in the ECoG in hypothermic animals compared to the hypothermic baseline, whereas the ECoG in normothermic animals showed a marked decrease in frequency, amplitude and total spectral power. We conclude that mild hypothermia produces an arousal-like ECoG activity with marked frequency shift to alpha activity and a change from high to low voltage activity. Furthermore, the hypothermic brain showed a preserved neuronal function at moderate stages of ICP. Obviously, hypothermia improves the functional tolerance of the brain to impaired oxygen supply.

Animals↗
PubMed1999

The VATAM guidelines.

Evaluation and assessment of the impact of information and communication technology in medicine is gaining interest. Unfortunately, till now there were no agreed upon approaches. The objective of the VATAM project is to develop guidelines that will assist assessors to set-up and execute studies. This paper describes the background of the VATAM project and provides an account of the current state of the guidelines. It concludes with an indication of the developments that will take place in the short term to further elaborate the guidelines and some considerations for consolidation of VATAM's results.

Databases, Factual↗
PubMed1999

Inventory of validation approaches in selected health telematics projects.

This paper presents the results from an inventory of validation approaches and methodologies which have been used in selected health telematics projects. The inventory was performed in the VATAM Validation of Telematic Applications in Medicine project, HC1115HC. The purpose of the inventory was to analyse the methodologies and their application assumptions in order to identify possibilities for harmonization and consolidation. The inventory was performed using five validation dimensions: IT-development; quality; user; technology assessment and marketing. The inventory results show that possibilities exist to synthesise methodologies and to provide practical guidance and support for projects that are developing health telematics applications. All stakeholders in health telematics projects, i.e. users, health care decision-makers, developers, suppliers and IT-industries, can benefit from practical validation guidelines and support for validation when guidelines are represented in a usable, easy to access and informative way.

Humans↗
PubMed1999

Receptor density as a factor governing the efficacy of the dopamine D4 receptor ligands, L-745,870 and U-101958 at human recombinant D4.4 receptors expressed in CHO cells.

1. The relationships between the density of dopamine D4.4 receptors and the agonist efficacies of L-745,870 (3-(4-[4-chlorophhenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2, 3-b]pyridine) and U-101958 ((1-benzyl-piperidin-4-yl)-(3-isopropoxy-pyridin-2-yl)-methyl-a min e) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. 2. In CHO cells expressing D4.4 receptors (CHO/D4 cells), dopamine inhibited forskolin-stimulated cyclic AMP accumulation (Emax 56+/-1% inhibition, pEC50 7.4+/-0.1, n=10). U-101958 behaved as a partial agonist (39+/-7% the efficacy of dopamine, pEC50 8.1+/-0.3, n=4), whereas L-745,870 had no detectable agonist effect. 3. Receptor density, as estimated by [3H]-spiperone saturation binding was 240+/-30 fmol mg-1 protein (n=8) in CHO/D4 cell homogenates. It reached 560+/-150 (n=6), 1000+/-190 (n=4) and 840+/-120 (n=4) fmol mg-1 protein after treatment with sodium butyrate (5 mM) for 6, 18 and 48 h, respectively. 4. The increase in receptor density was associated with a gradual enhancement of the agonist effects (increased Emax and pEC50 values) of dopamine. The efficacy of U-101958 (relative to dopamine) doubled and L-745,870 was turned into a partial agonist (efficacy 49% relative to dopamine, pEC50 8. 6+/-0.2, n=6, after 48 h treatment with sodium butyrate). These agonist effects of U-101958 and L-745,870 could be antagonized by spiperone (0.1 microM) but not by raclopride (10 microM). 5. The results show that U-101958 and L-745,870 are partial agonists at human dopamine D4.4 receptors expressed in CHO cells. Their efficacy is governed by receptor density. Agonist effects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels.

Aminopyridines↗
PubMed1999

Brain somatostatin: a candidate inhibitory role in seizures and epileptogenesis.

Recent evidence shows that neuropeptide expression in the CNS is markedly affected by seizure activity, particularly in the limbic system. Changes in neuropeptides in specific neuronal populations depend on the type and intensity of seizures and on their chronic sequelae (i.e. neurodegeneration and spontaneous convulsions). This paper reviews the effects of seizures on somatostatin-containing neurons, somatostatin mRNA and immunoreactivity, the release of this peptide and its receptor subtypes in the CNS. Differences between kindling and status epilepticus in rats are emphasized and discussed in the light of an inhibitory role of somatostatin on hippocampal excitability. Pharmacological studies show that somatostatin affects electrophysiological properties of neurons, modulates classical neurotransmission and has anticonvulsant properties in experimental models of seizures. This peptidergic system may be an interesting target for pharmacological attempts to control pathological hyperactivity in neurons, thus providing new directions for the development of novel anticonvulsant treatments.

Animals↗
PubMed1999

A piglet model for evaluation of cerebral blood flow and brain oxidative metabolism during gradual cerebral perfusion pressure decrease.

A piglet model was developed to study the effect of epidural volume expansion on cerebral perfusion pressure (CPP) by stepwise elevating intracranial pressure (ICP). Mean arterial blood pressure (ABP) was strictly maintained using an extracorporeal ABP controller. Two-week-old piglets (n = 10) were studied by surgically placing an epidural balloon over the right parietal region and gradually increasing the inflation to increase ICP to 25, 35 and 45 mm Hg, maintaining each pressure level for 30 min. Regional cerebral blood flow was measured using the colored microsphere technique, and cerebral oxygen delivery and cerebral metabolic rate of oxygen were calculated at baseline conditions and after reaching ICP levels of 25, 35 and 45 mm Hg. The results showed that this model of epidural volume expansion reproducibly reduces CPP to 70, 50 and 33% of baseline CPP values with elevation of ICP, and that the physiological variables remained stable throughout each increase in ICP. We conclude that the model simulates the effects of an acute intracranial focal mass expansion and is well suited for the evaluation of different therapeutical strategies for increased ICP in newborns and infants.

Animals↗
PubMed1998

Design, synthesis, and biological activities of potent and selective somatostatin analogues incorporating novel peptoid residues.

We report the synthesis, bioactivity, and structure-activity relationship studies of compounds related to the Merck cyclic hexapeptide c[Pro6-Phe7-d-Trp8-Lys9-Thr10-Phe11], L-363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with arylalkyl peptoid residues. We present a novel approach utilizing beta-methyl chiral substitutions to constrain the peptoid side-chain conformation. Our studies led to molecules which show potent binding and increased selectivity to the hsst2 receptor (weaker binding to the hsst3 and hsst5 receptors compared to L-363, 301). In vivo, these peptoid analogues selectively inhibit the release of growth hormone but have no effect on the inhibition of insulin. The biological assays which include binding to five recombinant human somatostatin receptors carried out in two independent laboratories and in vivo inhibition of growth hormone and insulin provide insight into the relationship between structure and biological activity of somatostatin analogues. Our results have important implications for the study of other peptide hormones and neurotransmitters.

Animals↗
PubMed1998

[125I][Tyr3]octreotide labels human somatostatin sst2 and sst5 receptors.

Human somatostatin (somatotropin release inhibiting factor = SRIF) receptor subtypes sst2 and sst5 were stably expressed in Chinese hamster lung fibroblast (CCL39) cells. [125I][Tyr3]octreotide labelled with high affinity and in a saturable manner both sst2 (pKd = 9.89+/-0.02, Bmax = 210+/-10 fmol/mg, n = 3) and sst5 sites (pKd = 9.64+/-0.04, Bmax = 920+/-170 fmol/mg, n = 3). The pharmacological profile of sst2 sites established in CCL39 cells using SRIF and various peptide analogues was very similar to that described previously in CHO cells and in human cortex: SRIF14 = SRIF28 > or = seglitide > BIM 23014 = RC 160 > octreotide > CGP 23996 > or = L362,855 > BIM 23052 > L361,301 = cortistatin14 > BIM 23030 > BIM 23056 > cycloantagonist SA. However, peptides classically perceived as sst2 receptor selective (e.g., seglitide, octreotide, vapreotide) showed also high affinity for human sst5 receptors labelled with [125I][Tyr3]octreotide: SRIF28 > seglitide > SRIF14 > L361,301 = octreotide > cortistatin14 = BIM 23014 = BIM 23052 > L362,855 = RC160 > CGP 23996 > BIM 23056 > cycloantagonist SA > BIM 23030. Further radioligand binding studies were performed with [Leu8,D-Trp22,125I-Tyr25]SRIF28 ([125I]LTT-SRIF28) and [125I]CGP 23996. At sst2 receptors, Bmax values determined with [125I][Tyr3]octreotide, [125I]LTT-SRIF28 and [125I]CGP 23996 were in the same range (180-370 fmol/mg). 5'-Guanylyl-imidodiphosphate (GppNHp) displaced all three radioligands to the same extent (85%) and the pharmacological profiles were superimposable. By contrast, at sst5 receptors Bmax values were very different: [125I][Tyr3]octreotide (920 fmol/mg), [125I]CGP 23996 (3530 fmol/mg) and [125I]LTT-SRIF28 (6950 fmol/mg). GppNHp affected [125I][Tyr3]octreotide more than [125I]CGP 23996 binding, whereas [125I]LTT-SRIF28 was much less affected. In addition, the affinity values determined in competition experiments at sst5 receptors, varied markedly; whereas SRIF14, cortistatin14 and SRIF28 showed 2-, 4- and 8-fold differences in affinity at sst5 receptors labelled with [125I][Tyr3]octreotide and [125I]LTT-SRIF28 compounds such as RC160, L363,301, L362,855, octreotide or CGP 23996 showed between 42- and 123-fold lower affinity when sst5 sites were labelled with [125I]LTT-SRIF28. The present data suggest caution to be used when comparing affinity profiles determined in binding studies using different radioligands. In addition, the present results suggest that effects produced by octreotide and related short chain SRIF analogues on hormone release, modulation of tumour growth and central effects may be mediated by either sst2 and/or sst5 receptors.

Animals↗
PubMed1998

[125I]Tyr10-cortistatin14 labels all five somatostatin receptors.

The recently cloned rat preprocortistatin, which shows homology to the preprosomatostatin peptide, is thought to be enzymatically cleaved to cortistatin14 (CST14) similarly to somatostatin14 (SRIF14). High structural similarity of cortistatin14 compared to SRIF14 suggested binding properties to somatostatin receptors similar to SRIF14. In the present study, we expressed stably the five human somatostatin receptor subtypes (hsst1-hsst5) in CCL39 cells (Chinese hamster lung fibroblast cells). The receptors were labelled with an iodinated analogue of CST14 ([125I]Tyr10)-cortistatin14, [125I]Tyr10-CST) to establish the pharmacological profile of hsst1-hsst5 sites labelled with [125I]Tyr10-CST. In parallel, [Leu8,D-Trp22,125I-Tyr25]-SRIF28 ([125I]LTT-SRIF28) was used as a control at the five recombinant SRIF receptors stably expressed in CCL39 cells. High affinity [125I]Tyr10-CST binding could be demonstrated to all five recombinant somatostatin receptor subtypes. The pKd (-log mol/l) and Bmax values (fmol/mg) for hsst1-5 receptors were: 10.02+/-0.04, 220+/-30; 9.45+/-0.09, 340+/-70; 10.06+/-0.11, 340+/-50; 9.67+/-0.14, 340+/-110 and 10.33+/-0.03, 5630+/-1330, respectively. The pharmacological profiles determined with [125I]Tyr10-CST and [125I]LTT-SRIF28 were very similar at every receptor studied. These data suggest that cortistatin and somatostatin have similar high affinity for SRIF receptors. None of the receptors showed marked selectivity for either CST14/CST17 or the somatostatins. In conclusion, the data show that cortistatin and somatostatin have very similar high affinity to all five recombinant somatostatin receptors. It remains to be seen whether there are specific receptors which bind only somatostatins or cortistatins.

Amino Acid Sequence↗
PubMed1998

The agonist activities of the putative antipsychotic agents, L-745,870 and U-101958 in HEK293 cells expressing the human dopamine D4.4 receptor.

1. Dopamine D4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced. 2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D4.4 receptor (HEK293/D4 cells). [3H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3. [3H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D4 cell homogenates (Bmax 505+/-90 fmol mg(-1) protein, pK(D) 9.5+/-0.1, n=3). Inhibition of specific [3H]-spiperone binding was observed with spiperone (pKi 9.6+/-0.1, n=3), clozapine (pKi 7.4+/-0.1, n=4), L-745,870 (pKi 8.5+/-0.1, n=3) and U-101958 (pKi 8.9+/-0.1, n=3). By contrast, raclopride was very weak (pKi < 5, n=3). 4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent fashion (Emax 71+/-2% inhibition of forskolin-stimulated levels, pEC50 8.7+/-0.1, n=10). This effect was mimicked by the dopamine D2-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). 5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC50) values of L-745,870 and U-101958 were 9.0+/-0.2 (n=4) and 8.7+/-0.3 (n=3), consistent with pKi values determined in radioligand binding studies. 6. Dopamine, L-745,870 and U-101958 (up to 1 microM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells. 7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pK(B) 7.1), but not by raclopride (pK(B) < 5). None of these antagonists had any significant agonist activity at concentrations up to 10 microM. 8. These results show that the putative dopamine D4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.

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