Lin-Jun Ge

ISOLATION OF RELATIVELY LARGE AMOUNTS OF ENDOMORPHIN-1 AND ENDOMORPHIN-2 FROM HUMAN BRAIN CORTEX

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were previously isolated from bovine brain. Both peptides showed the greatest selectivity and affinity for the mu opiate receptor of any endogenous substance found to date and may serve as natural ligands for the mu-opiate receptor. We have purified them from the fronto-parietal cortex of human brain tissue by solid phase extraction and high performance liquid chromatography. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against endomorphin-1. The isolated endomorphins showed full biological activity. The tetrapeptides were found in human brain in much higher amounts than in bovine frontal cortex.

Endomorphins: Novel Endogenous μ‐Opiate Receptor Agonists in Regions of High μ‐Opiate Receptor Density

Abstract: Endomorphin‐1 (Tyr‐Pro‐Trp‐Phe‐NH2, EM‐1) and endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH2, EM‐2) are peptides recently isolated from brain that show the highest affinity and selectivity for the m (morphine) opiate receptor of all the known endogenous opioids. The endomorphins have potent analgesic and gastrointestinal effects. At the cellular level, they activate G‐proteins (35S‐GTP γ‐S binding) and inhibit calcium currents. Support for their role as endogenous ligands for the μ‐opiate receptor includes their localization by radioimmunoassay and immunocytochemistry in central nervous system regions of high μ receptor density. Intense EM‐2 immunoreactivity is present in the terminal regions of primary afferent neurons in the dorsal horn of the spinal cord and in the medulla near high densities of μ receptors. Chemical (capsaicin) and surgical (rhizotomy) disruption of nociceptive primary afferent neurons depletes the immunoreactivity, implicating the primary afferents as the source of EM‐2. Thus, EM‐2 is well‐positioned to serve as an endogenous modulator of pain in its earliest stages of perception. In contrast to EM‐2, which is more prevalent in the spinal cord and lower brainstem, EM‐1 is more widely and densely distributed throughout the brain than EM‐2. The distribution is consistent with a role for the peptides in the modulation of diverse functions, including autonomic, neuroendocrine, and reward functions as well as modulation of responses to pain and stress.

Endomorphin 1 and 2, endogenous ligands for the μ-opioid receptor, decrease cardiac output, and total peripheral resistance in the rat

Endomorphin 1 and 2 are recently discovered endogenous ligands for the mu-opioid receptor. In the present study, responses to intravenous administration of endomorphin 1 and 2 were investigated in the systemic vascular bed of the rat. Endomorphin 1 and 2 induced dose-related decreases in systemic arterial pressure when injected in doses of 10-100 nmol/kg i.v.. The decreases in systemic arterial pressure in response to endomorphin 1 and 2 were associated with significant decreases in heart rate, cardiac output, and total peripheral resistance. The endogenous ligand for the ORL1 receptor, nociceptin/OFQ had similar effects on systemic arterial pressure, heart rate, cardiac output, and total peripheral resistance in the rat. Injections of isoproterenol (1 microgram/kg i.v.) and calcitonin gene-related peptide (CGRP; 0.3 nmol/kg i.v.), decreased systemic arterial pressure and total peripheral resistance. However these decreases in arterial pressure were associated with increases in heart rate and cardiac output. The results of the present study demonstrate that the endomorphin peptides have significant vasodilator activity in the systemic vascular bed of the rat and show that this response is associated with a decrease in heart rate and cardiac output.

MU, DELTA, AND KAPPA OPIATE RECEPTOR BINDING OF TYR-MIF-1 AND OF TYR-W-MIF-1, ITS ACTIVE FRAGMENTS, AND TWO POTENT ANALOGS

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 microM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (Ki = 71 nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH2, show high affinity as well as potent analgesic activity.

Endomorphin 1 and 2, endogenous μ-opioid agonists, decrease systemic arterial pressure in the rat

The endogenous opioid peptides, endomorphin 1 and 2, are newly isolated, potent, and selective mu-opioid receptor agonists. In the present study, responses to endomorphin 1 and 2 were investigated in the systemic vascular bed of the rat. Endomorphin 1 and 2 induced dose-related decreases in systemic arterial pressure when injected in doses of 1-30 nmol/kg i.v. In terms of relative vasodepressor activity, endomorphin 1 and 2 were approximately equipotent with each other and with the ORL1 ligand, nociceptin (orphanin FQ), and were about 10-fold more potent than met-enkephalin in decreasing systemic arterial pressure. Vasodepressor responses to endomorphin 1 and 2 and met-enkephalin, but not to nociceptin, were inhibited by the opioid receptor antagonist, naloxone. These results demonstrate that endomorphin 1 and 2 produce significant naloxone-sensitive decreases in systemic arterial pressure.

Chronic, but not acute, administration of morphine alters antiopiate (Tyr-MIF-1) binding sites in rat brain

Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (Tyr-MIF-1) as well as opiate binding sites in rat brain

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.

Binding of Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) and related peptides to μ1 and μ2 opiate receptors

Abstract Two endogenous brain peptides (Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH 2 ) and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH 2 )), a cyclized analog and two fragments of Tyr-W-MIF-1, and hemorphin (Tyr-Pro-Trp-Thr) were tested for binding to μ 1 and μ 2 opiate receptors. All these peptides bound to both μ 1 and μ 2 sites in assays optimized to discriminate these subtypes of the μ opiate receptor in membranes from bovine thalamus. The cyclized analog of Tyr-W-MIF-1, previously shown to have potency near that of Tyr- d -Ala-Gly- N -MePhe-Gly-ol (DAMGO) and morphine in producing analgesia after intracerebroventricular (i.c.v.) injection, bound to μ 1 and μ 2 sites with affinities similar to those of (DAMGO). Tyr-W-MIF-1, previously shown to induce analgesia after i.c.v. injection but with much higher potency after intrathecal (i.t.) injection, also bound to both μ 1 and μ 2 sites with an affinity between that of morphiceptin and hemorphin. Although the highest ratios of K i s for μ 2 / μ 1 were shown by hemorphin, Tyr-MIF-1, and Tyr-W-MIF-1, none of the compounds were significantly different in selectivity. The results indicate that the relatively lower potency of Tyr-W-MIF-1 after i.c.v., compared with i.t. injection, is not due to a lack of binding to μ 1 sites. They suggest that it has relatively high efficacy at μ 2 , but low efficacy at μ 1 sites, a possibility that might explain some of the novel properties of these peptides.

Novel peptides and mechanisms of opiate tolerance

Abstract We have used two diverse approaches for studying cellular and tissue effects of chronic exposure to opiates. First, receptor regulation by agonists and antagonists was characterized in a human neuroblastoma cell line, SH-SY5Y. Morphine down-regulated mu and delta opiate receptors. Mu receptor down-regulation was selectively blocked by the mu antagonist CTOP and delta receptor changes by the delta antagonist ICI 174864. Naloxone up-regulated opiate receptors in these cells, as it does in brain. Thus, SH-SY5Y cells provided a neuronal model for studying selective mu and delta receptor regulation in response to agonists and antagonists. In a second approach, we have shown that peptides recently isolated from brain in our laboratory (Tyr-MIF-1 and Tyr-W-MIF-1) can act as opiate agonists in several test paradigms, but as antagonists under specific circumstances in these test systems. These include chronic exposure to morphine or reduction of receptor reserve by alkylating agents. These peptides may serve as prototypes for endogenous compounds that act as agonists in the naive state and as antagonists in the tolerant state.

Opiate receptor binding, guinea pig ileum activity, and prolonged analgesia induced by the brain peptide Tyr-W-MIF-1 and two potent analogs

Abstract Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH 2 ) is a peptide isolated from human and bovine brain with opiate activity. Two synthetic analogs of Tyr-W-MIF-1 were compared with the parent compound, DAMGO and Tyr-MIF-1 for (a) binding to opiate receptors (b) activity in the guinea pig ileum assay, and (c) analgesic activity after ICV administration to the rat. All compounds tested showed preferential binding to mu receptors. Tyr-MIF-1 showed relatively low affinity, but high selectivity (∼400-fold selectivity for mu over delta and more than 500-fold selective for mu over kappa sites). Tyr-W-MIF-1, which induces prolonged analgesia after ICV administration, showed affinity for mu sites that was 10-fold higher than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa sites. Two cyclized analogs of Tyr-W-MIF-1 approached the potency of DAMGO in several assays. The analogs showed 50- and 20-fold increases in affinity (IC 50 of 6 and 18 nM) for the mu site relative to Tyr-W-MIF-1, with 50-fold selectivity for mu over delta sites, and lack of binding to non-opiate Tyr-MIF-1 sites. The analogs were active in the guinea pig ileum assay with IC 50 s of 10 and 30 nM. Dose-dependent analgesia after ICV administration of one of the analogs persisted for about an hr after 1 μg, for 3 hr after 25 μg, and for more than 6 hr after 100 μg. For both analogs, analgesia after 1 μg was similar to that seen with DAMGO. Thus, an endogenous brain peptide (Tyr-W-MIF-1) that does not contain the sequence Tyr-Gly-Gly, together with its structural analogs, can provide a range of selectivity patterns for binding to opiate receptors and potent opiate agonist activity.