DPDPE Research Articles

Tonic and reflex control of the cardio-respiratory system by neurons in the ventral medulla

To investigate the channels and neurotransmitters in the ventrolateral medulla (VLM) oblongata that are responsible for the maintenance of sympathetic tone and cardio-respiratory reflex regulation. Microinjections of excitant amino acid (glutamate, 100 nl, 100 mM), calcium channel blockers, agonists and antagonists were made throughout the VLM in anaesthetized rats. Arterial blood pressure, sympathetic nerve activity and phrenic nerve discharge was recorded. Responses to activation of the baroreceptor, somatosympathetic, and chemoreceptor reflexes were recorded. Injection sites were marked with albumin-colloidal gold or methylene blue. At the end of recording, brains were removed, fixed, sectioned and stained for gold spots, and counterstained to localize injection sites. Bilateral microinjection of calcium channel antagonists into the rostral VLM (RVLM) had little effect on arterial blood pressure with the exception of Ni++ (an agent that blocks low voltage T-type calcium channels). Ni++ caused a profound fall in arterial blood pressure and sympathetic nerve activity, but did not affect reflex function. By contrast the high voltage channel (P, Q, L, N) blockers increased blood pressure and sympathetic activity following injection into the caudal VLM (CVLM). The agents applied were 8-OHDPAT; 5HT1a agonist, DAMGO; mu opioid agonist, DPDPE; delta opioid agonist and sar9-substance P; substance P agonist. In addition, the effect of changing the inhaled concentration of carbon dioxide was tested. In all cases except injection DAMGO, there was an attenuation of the somato-sympathetic reflex. DAMGO attenuated the baroreceptor reflex. None of these agents affected the chemoreceptor reflex. The data suggest that tonic maintenance of sympathetic activity is mediated, at least in part, by a Ni++ sensitive conductance in RVLM and that the neurochemical heterogeneity found within the inputs to sympathoexcitatory and inhibitory neurons in the RVLM has functional significance, with different neurotransmitters affecting cardiorespiratory reflexes differentially. Further functional studies are required to determine the precise connectivity between different neuronal populations that are subserving different functions in the RVLM.

A Role for the Distal Carboxyl Tails in Generating the Novel Pharmacology and G Protein Activation Profile of μ and δ Opioid Receptor Hetero-oligomers

Opioid receptor pharmacology in vivo has predicted a greater number of receptor subtypes than explained by the profiles of the three cloned opioid receptors, and the functional dependence of the receptors on each other shown in gene-deleted animal models remains unexplained. One mechanism for such findings is the generation of novel signaling complexes by receptor hetero-oligomerization, which we previously showed results in significantly different pharmacology for mu and delta receptor hetero-oligomers compared with the individual receptors. In the present study, we show that deltorphin-II is a fully functional agonist of the mu-delta heteromer, which induced desensitization and inhibited adenylyl cyclase through a pertussis toxin-insensitive G protein. Activation of the mu-delta receptor heteromer resulted in preferential activation of Galpha(z), illustrated by incorporation of GTPgamma(35)S, whereas activation of the individually expressed mu and delta receptors preferentially activated Galpha(i). The unique pharmacology of the mu-delta heteromer was dependent on the reciprocal involvement of the distal carboxyl tails of both receptors, so that truncation of the distal mu receptor carboxyl tail modified the delta-selective ligand-binding pocket, and truncation of the delta receptor distal carboxyl tail modified the mu-selective binding pocket. The distal carboxyl tails of both receptors also had a significant role in receptor interaction, as evidenced by the reduced ability to co-immunoprecipitate when the carboxyl tails were truncated. The interaction between mu and delta receptors occurred constitutively when the receptors were co-expressed, but did not occur when receptor expression was temporally separated, indicating that the hetero-oligomers were generated by a co-translational mechanism.

Characterization of the bioactive form of linear peptide antagonists at the omega-opioid receptor.

The stereochemical requirements for omega-opioid receptor binding of a series of linear peptide antagonists with a novel conformationally restricted Phe analogue (Tic) as a second residue were examined by using a variety of computational chemistry methods. The omega-opioid receptor analogues with significant affinity, Tyr-Tic-NH2(TI-NH2), Tyr-Tic-Phe-OH(TIP), Tyr-Tic-Phe-NH2(TIP-NH2), Tyr-Tic-Phe-Phe-OH(TIPP), Tyr-Tic-Phe-Phe-NH2)(TIPP-NH2), and the low affinity omega-opioid peptides Tyr-Pro-Phe-Pro-NH2(morphiceptin) and Tyr-Phe-Phe-Phe-NH2 (TPPP-NH2), were included in this study. The conformational profiles of these peptides were obtained by consecutive cycles of high and low temperature molecular dynamic stimulations, coupled to molecular mechanical energy minimization carried out until no new conformational minima were obtained. Comparing the results for TPPP-NH2 and TIPP-NH2, the presence of the conformationally restricted Tic residue did not greatly reduce the number of unique low energy conformations, but did allow low energy conformers involving cis bonds between the first two residues. The conformational libraries of these peptides were examined for their ability to satisfy the three key ligand components for receptor recognition already identified by previous studies of high affinity cyclic (Tyr1-D-Pen2-Gly3-Phe4-D-Pen5) enkephalin (DPDPE) type agonists: a protonated amine group, an aromatic ring, and a lipophilic moiety in a specific geometric arrangement. Two types of conformations common to the five high omega-opioid affinity L-Tic analogues were found that satisfied these requirements, one with a cis and the other with a trans peptide bond between the Tyr1 and Tic2 residues. Moreover, both the Tic2 and Phe3 residues could mimic the hydrophobic interactions with the receptor of the Phe4 moiety in the cyclic DPDPE type agonists, consistent with the appreciable affinity of both di- and tripeptides. The low omega-opioid receptor affinity of morphiceptin can be understood as the result of conformational preferences that prevent the fulfillment of this pharmacophore for recognition.

Modulation of non-adrenergic non-cholinergic inhibitory transmission in rat duodenum: role of opiates and 5-hydroxytryptamine.

In rat duodenal segments in vitro, electrical field stimulation induced a TTX-sensitive relaxation in the presence of atropine and guanethidine. A correlation between the amplitude of the evoked response and stimulus frequency was observed. Opioid peptides DAGO, DPDPE and DYN caused a dose-dependent increase in the amplitude of the response to EFS. Naloxone shifted to the right the dose-response curves for each opioid peptide significantly enhancing the ED50 values. The amplitude of the response to EFS was enhanced, dose-dependently, also in the presence of 5-HT. Such an effect induced by 5-HT was prevented by 5-HT receptor desensitization, but persisted unchanged after naloxone pretreatment. Opioids failed to affect the response to EFS after 5-HT receptor desensitization. Results suggest that in rat duodenum opioids modulate NANC inhibitory neurotransmission, indirectly the release of 5-HT.