Rubsicolins are naturally occurring G-protein-biased delta opioid receptor peptides

The impact that β-arrestin proteins have on G-protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of β-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit β-arrestin, activating only the canonical G-protein pathway. Similar G-protein biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G-protein-biased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G-protein signaling pathways with minimal β-arrestin recruitment. We find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G-protein signaling at δ opioid receptors. However, unlike the naturally occurring δ opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit β-arrestin 2 and have undectable recruitment of β-arrestin 1 at the δ opioid receptor.

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na+-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H+/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na+-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala2,d-Leu5]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na+ dependent, with more than 2 Na+ involved in the uptake process. In contrast, DADLE uptake was only partially Na+ dependent. The uptake of both peptides was also influenced by H+ and Cl−, although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na+ dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9–17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.

Behavioral Consequences of Delta-Opioid Receptor Activation in the Periaqueductal Gray of Morphine Tolerant Rats

Chronic morphine administration shifts delta-opioid receptors (DORs) from the cytoplasm to the plasma membrane. Given that microinjection of morphine into the PAG produces antinociception, it is hypothesized that the movement of DORs to the membrane will allow antinociception to the DOR agonist deltorphin II as a way to compensate for morphine tolerance. Tolerance was induced by twice daily injections of morphine (5, 10, or 20 mg/kg, subcutaneous) for 3.5 days. Microinjection of deltorphin into the vPAG 6 hours after the last morphine injection produced a mild antinociception that did not vary in a consistent manner across morphine pretreatment doses or nociceptive tests. In contrast, deltorphin caused a decrease in activity in morphine tolerant rats that was associated with lying in the cage. The decrease in activity and change in behavior indicate that chronic morphine administration alters DORs in the vPAG. However, activation of these receptors does not appear to compensate for the decrease in antinociception caused by morphine tolerance.

Opioid Receptors on Bulbospinal Respiratory Neurons Are Not Activated During Neuronal Depression by Clinically Relevant Opioid Concentrations

Opioids depress the activity of brain stem respiratory-related neurons, but it is not resolved whether the mechanism at clinical concentrations consists of direct neuronal effects or network effects. We performed extracellular recordings of discharge activity of single respiratory neurons in the caudal ventral respiratory group of decerebrate dogs, which were premotor neurons with a likelihood of 90%. We used multibarrel glass microelectrodes, which allowed concomitant highly localized picoejection of opioid receptor agonists or antagonists onto the neuron. Picoejection of the μ receptor agonist [d-Ala2, N-Me-phe4, gly-ol5]-enkephalin (DAMGO, 1 mM) decreased the peak discharge frequency (mean ± SD) of expiratory neurons to 68 ± 22% ( n = 12), the δ1 agonist d-Pen2,5-enkephalin (DPDPE, 1 mM) to 95 ± 11% ( n = 15), and δ2 receptor agonist [d-Ala2] deltorphin-II to 86 ± 17% (1 mM, n = 15). The corresponding values for inspiratory neurons were: 64 ± 12% ( n = 11), 48 ± 30% ( n = 12), and 75 ± 15% ( n = 11), respectively. Naloxone fully reversed these effects. Picoejection of morphine (0.01–1 mM) depressed most neurons in a concentration dependent fashion to maximally 63% ( n = 27). Picoejection of remifentanil (240–480 nM) did not cause any significant depression of inspiratory ( n = 11) or expiratory neurons ( n = 9). 4. Intravenous remifentanil (0.2–0.6 μg·kg−1·min−1) decreased neuronal peak discharge frequency to 60 ± 12% (inspiratory, n = 7) and 58 ± 11% (expiratory, n = 11). However, local picoejection of naloxone did not reverse the neuronal depression. Our data suggest that μ, δ1, and δ2 receptors are present on canine respiratory premotor neurons. Clinical concentrations of morphine and remifentanil caused no local depression. This lack of effect and the inability of local naloxone to reverse the neuronal depression by intravenous remifentanil suggest that clinical concentrations of opioids produce their depressive effects on mechanisms upstream from respiratory bulbospinal premotor neurons.