Hartmut Berger

Corticotropin-releasing hormone (CRH) receptors in the mesenteric small arteries of rats resemble the (2)-subtype☆

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

Pharmacological characterisation of [(pX)Phe4]nociceptin(1-13)amide analogues. 1. In vitro studies.

Abstract. Phe4 in the nociceptin (NC) sequence has been identified as the most critical residue for receptor interaction. In the present study, we investigated the pharmacological activity of a series of NC(1-13)NH2 analogues, in which the hydrogen atom in the para position of Phe4 was substituted with F, NO2, CN, Cl, Br, I, CH3, OH or NH2.In receptor binding studies, performed using CHO cells expressing the recombinant human NC receptor (CHOhOP4) and in rat cerebral cortex membranes, [(pF)Phe4]NC(1-13)NH2, [(pNO2)Phe4]NC(1-13)NH2, and [(pCN)Phe4]NC(1-13)NH2 displayed higher affinity than NC(1-13)NH2. The affinity of [(pCl)Phe4]NC(1-13)NH2 was essentially identical to that of NC(1-13)NH2, while the remaining compounds displayed reduced affinity. In a series of functional assays (stimulation of GTPγS binding in CHOhOP4 cells and rat cerebral cortex membranes and inhibition of cAMP accumulation in CHOhOP4 cells), the para substituted analogues behaved as full agonists (with the exception of [(pOH)Phe4]NC(1-13)NH2 which acted as a partial agonist in the GTPγS binding assays) with the following rank order potency:

Response of the glycolysis of human erythrocytes to the transition from the oxygenated to the deoxygenated state at constant intracellular pH

{\rm pF = pNO}_{\rm 2} \ge {\rm pCN} \ge {\rm NC = NC(1 - 13)NH}_{\rm 2} {\rm = pCl} \ge {\rm pBr > pI = pCH}_{\rm 3} {\rm > pOH > pNH}_{\rm 2}

Evidence for extensive and non-specific translocation of oligopeptides across plasma membranes of mammalian cells

[(pF)Phe4]NC(1-13)NH2 and [(pNO2)Phe4]NC(1-13)NH2 were either inactive or displayed micromolar potencies in cAMP accumulation experiments performed on cells expressing classical opioid receptors. All compounds were full agonists in isolated tissues from various species (guinea pig ileum, mouse colon and mouse/rat vas deferens) with the exception of [(pOH)Phe4]NC(1-13)NH2 which displayed partial agonist/weak antagonist activities. The rank order of potency was similar to that found in the other assays. The effects of all analogues were not modified by naloxone. The selective OP4 receptor antagonist [Nphe1]NC(1-13)NH2, tested in all preparations against one or both of the highly potent derivatives [(pF)Phe4]NC(1-13)NH2 and [(pNO2)Phe4]NC(1-13)NH2, showed pA2 values similar to those found against NC, the pA2 in the GTPγS binding/rat cerebral cortex assay being much higher (ca. 7.5) than in the other functional assays (ca. 6).This study further supports the notion that Phe4 of NC is the critical residue for receptor occupation and activation. Moreover, as part of this study, we have identified two novel, highly potent and selective agonists for the OP4 receptor, [(pF)Phe4]NC(1-13)NH2 and [(pNO2)Phe4]NC(1-13)NH2.

Inhibitory action of nociceptin/orphanin FQ on functionally different thalamic neurons in urethane-anaesthetized rats

The time course of the rate of the glycolysis of human erythrocytes and of some metabolites were determined before and after rapid deoxygenation at constant intracellular pH. For this purpose stripped deoxygenated haemoglobin was used as a rapid oxygen acceptor. Deoxygenation causes an increase of the glycolytic rate by 26%. Glucose 6-phosphate is decreased while the adenine nucleotides and 2,3-bisphosphoglycerate remain constant. Fructose 1,6-bisphosphate and the triose phosphates decrease transiently before rising. The data can be explained by increased binding of phosphocompounds to deoxygenated as compared with oxygenated haemoglobin. Thereby the control enzymes hexokinase and phosphofructokinase are influenced. It is concluded that under physiological conditions changes in the oxygenation state of haemoglobin per se alter the glycolytic rate.

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats

After exposure of bovine aortic endothelial cells to various small peptides (tetra- to undeca-mer), extensive transport of the peptides across the plasma membrane was observed in the concentration range 10(-7) to 10(-2) M. The observed transport events, which contradict the generally anticipated poor permeability of peptides across plasma membranes, exhibited high complexity and showed no saturability up to a concentration of 10(-2) M. Evidence was found for the involvement of mdrp-like transporters as well as of energy-independent facilitated diffusion events. The peptide levels within the cells approximated those of the incubation solution within 30 min, indicating high capacity and velocity for the involved transport processes. Correspondingly, preloaded cells exported about 80% of the internalized peptide within 5 min at 37 degrees C. Analogous results were found after peptide exposure to several other mammalian cell types, indicating a more general importance of the transport phenomena described here. Our findings contradict the prevailing opinion that the often observed lack of activity of externally administered peptides against their targets within intact cells is accounted for primarily by poor cellular uptake and point to export processes counteracting the uptake to be more important in this context.

Influence of Continuous Levels of Fentanyl in Rats on the μ-Opioid Receptor in the Central Nervous System

In this study we administered nociceptin/orphanin FQ (NC) ionotophoretically onto neurons located in functionally distinct thalamic structures of urethane‐anesthetized rats. Extracellular single unit recordings were made in the medial and lateral ventroposterior nucleus, posterior thalamic nucleus, zona incerta, lateral posterior nucleus, laterodorsal nucleus, ventrolateral nucleus and reticular nucleus. NC decreased the firing rate in 60% of thalamic neurons. This decrease in firing rate was accompanied by a significant reduction in the number of high threshold bursts. In about 20% of the neurons NC increased the firing rate. In most cells NC‐induced increases in discharge rate could be blocked by the GABAA receptor antagonists bicuculline and SR 95531. The NC receptor ligands [Phe1Ψ(CH2‐NH)Gly2] nociceptin(1‐13)NH2, Ac‐RYYRIK‐NH2 and [Nphe1]NC(1‐13)NH2 were also evaluated. All these peptides inhibited NC‐induced changes in firing rate. In addition, in some neurons where NC inhibited firing, [Nphe1]NC(1‐13)NH2 and Ac‐RYYRIK‐NH2 elicited per se an increase in firing rate, suggesting the existence of tonic innervation of thalamic neurons by NC‐containing fibres. In NC‐inhibited neurons nocistatin induced a significant increase in firing rate. The present study demonstrated that NC regulates various thalamic nuclei related not only to somatosensory, but also to the visual and motor functions.

Proteolytic inactivation of luteinizing hormone-releasing hormone (LHRH) by the whole rat ovary in vitro

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.

Proteolytic Degradation of Gonadotropin-Releasing Hormone (GnRH) by Rat Ovarian Fractions In Vitro

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.