Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury.

Reactive oxygen species (ROS) play a key role in promoting mitochondrial cytochrome c release and induction of apoptosis. ROS induce dissociation of cytochrome c from cardiolipin on the inner mitochondrial membrane (IMM), and cytochrome c may then be released via mitochondrial permeability transition (MPT)-dependent or MPT-independent mechanisms. We have developed peptide antioxidants that target the IMM, and we used them to investigate the role of ROS and MPT in cell death caused by t-butylhydroperoxide (tBHP) and 3-nitropropionic acid (3NP). The structural motif of these peptides centers on alternating aromatic and basic amino acid residues, with dimethyltyrosine providing scavenging properties. These peptide antioxidants are cell-permeable and concentrate 1000-fold in the IMM. They potently reduced intracellular ROS and cell death caused by tBHP in neuronal N(2)A cells (EC(50) in nm range). They also decreased mitochondrial ROS production, inhibited MPT and swelling, and prevented cytochrome c release induced by Ca(2+) in isolated mitochondria. In addition, they inhibited 3NP-induced MPT in isolated mitochondria and prevented mitochondrial depolarization in cells treated with 3NP. ROS and MPT have been implicated in myocardial stunning associated with reperfusion in ischemic hearts, and these peptide antioxidants potently improved contractile force in an ex vivo heart model. It is noteworthy that peptide analogs without dimethyltyrosine did not inhibit mitochondrial ROS generation or swelling and failed to prevent myocardial stunning. These results clearly demonstrate that overproduction of ROS underlies the cellular toxicity of tBHP and 3NP, and ROS mediate cytochrome c release via MPT. These IMM-targeted antioxidants may be very beneficial in the treatment of aging and diseases associated with oxidative stress.

Comparison of [Dmt1]DALDA and DAMGO in binding and G protein activation at mu, delta, and kappa opioid receptors.

[Dmt1]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt = 2',6'-dimethyltyrosine) binds with high affinity and selectivity to the mu opioid receptor and is a surprisingly potent and long-acting analgesic, especially after intrathecal administration. In an attempt to better understand the unique pharmacological profile of [Dmt1]DALDA, we have prepared [3H][Dmt1]DALDA and compared its binding properties with that of [3H]DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin). Kinetic studies revealed rapid association of [3H][Dmt1]DALDA when incubated with mouse brain membranes (K+1 = 0.155 nM(-1) min(-1)). Dissociation of [3H][Dmt1]DALDA was also rapid (K(-1) = 0.032 min(-1)) and indicated binding to a single site. [3H][Dmt1]DALDA binds with very high affinity to human mu opioid receptor (hMOR) (Kd = 0.199 nM), and Kd and Bmax were reduced by sodium but not Gpp(NH)p [guanosine 5'-(beta,gamma-imido)triphosphate]. Similar Kd values were obtained in brain and spinal cord tissues and SH-SY5Y cells. The hMOR:hDOR (human delta opioid receptor) selectivity of [Dmt1]DALDA ( approximately 10,000) is 8-fold higher than DAMGO. However, [Dmt1]DALDA is less selective than DAMGO against hKOR (human kappa opioid receptor) (26-versus 180-fold). The Ki values for a number of opioid ligands were generally higher when determined by competitive displacement binding against [3H][Dmt1]DALDA compared with [3H]DAMGO, with the exception of Dmt1-substituted peptide analogs. All Dmt1 analogs showed much higher affinity for the mu receptor than corresponding Tyr1 analogs. [35S]GTPgammaS (guanosine 5'-O -(3-[35S]thio)triphosphate) binding showed that [Dmt1]DALDA and DAMGO are full agonists at hMOR and hDOR but are only partial agonists at hKOR. The very high affinity and selectivity of [3H][Dmt1]DALDA for the mu receptor, together with its very low nonspecific binding (10-15%) and metabolic stability, make [3H][Dmt1]DALDA an ideal radioligand for labeling mu receptors.

Endogenous opioid peptides contribute to antinociceptive potency of intrathecal [Dmt1]DALDA.

[Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) is a dermorphin analog that shows high affinity and selectivity for the mu opioid receptor. The intrathecal potency of [Dmt(1)]DALDA far exceeded its affinity at mu receptors and suggests that other mechanisms must be involved in its action in the spinal cord. The affinity and selectivity of [Dmt(1)]DALDA was determined using cell membranes expressing cloned human mu, delta, and kappa opioid receptors. Competitive displacement binding with [(3)H][Dmt(1)]DALDA, [(3)H]DPDPE (H-Tyr-d-Pen-Gly-Phe-d-Pen), and [(3)H]U69,593 [(5alpha,7alpha,8beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4.5]dec-8-yl)-benzeneacetamide] revealed K(i) of 156 +/- 26 pM for mu opioid receptor (MOR), 1.67 +/- 0.04 microM for delta opioid receptor (DOR), and K(i) of 4.4 +/- 1.7 nM for kappa opioid receptor (KOR), respectively. [Dmt(1)]DALDA increased guanosine 5'-O-(3-[(35)S]thiotriphosphate) binding in MOR, DOR, and KOR membranes, with EC(50) being 17 (8.8-33) nM, 2 (1.2-3.2) microM, and 124 (15-1000) nM, respectively. Intrathecal [Dmt(1)]DALDA inhibited the tail-flick response in mice with ED(50) = 1.22 (0.59-2.34) pmol. Intrathecal administration of an antiserum against dynorphin A(1-17) or [Met(5)]enkephalin significantly attenuated the response to i.t. [Dmt(1)]DALDA, resulting in ED(50) of 6.2 (3.6-12.6) pmol and 6.6 (3.5-19.6) pmol, respectively. Neither antisera had any effect on the response to i.t. morphine. Intracerebroventricular (i.c.v.) [Dmt(1)]DALDA was not affected by previous i.c.v. administration of anti-Dyn or anti-ME. Pretreatment with norbinaltorphimine or naltriben also attenuated the antinociceptive response to i.t., but not i.c.v., [Dmt(1)]DALDA. These data suggest that i.t. [Dmt(1)]DALDA causes the release of dynorphin and [Met(5)]enkephalin-like substances that act at kappa and delta receptors, respectively, to contribute to the extraordinary potency of [Dmt(1)]DALDA.

Transcellular transport of a highly polar 3+ net charge opioid tetrapeptide.

Oligopeptides are generally thought to have poor permeability across biological membranes. Recent studies, however, suggest significant distribution of [Dmt1]DALDA (Dmt-D-Arg-Phe-Lys-NH2; Dmt is 2',6'-dimethyltyrosine), a 3+ net charge opioid peptide, to the brain and spinal cord after subcutaneous administration. Peptide transporters (PEPT1 and PEPT2) play a major role in the uptake of di- and tripeptides across cell membranes, but their ability to transport tetrapeptides is not clear. The purpose of this study was to determine whether [Dmt1]DALDA can translocate across Caco-2 cell monolayers and whether PEPT1 plays a role in the uptake process. Our results show that [3H][Dmt1]DALDA can readily translocate across Caco-2 cells, with a permeability coefficient estimated to be 1.24 x 10(-5) cm/s. When incubated with Caco-2 cells, [3H][Dmt1]DALDA was detected in cell lysates by 5 min. The internalization of [Dmt1]DALDA was confirmed visually with a fluorescent [Dmt1]DALDA analog (H-Dmt-D-Arg-Phe-dnsDap-NH2; dnsDap is beta-dansyl-L-alpha,beta-diaminopropionic acid). The uptake of [3H][Dmt1]DALDA was concentration-dependent but temperature- and pH-independent. Treatment with diethylpyrocarbonate (DEPC) inhibited [14C]glycine-sarcosine uptake but increased [3H][Dmt1]DALDA uptake 34-fold. These findings suggest that PEPT1 is not involved in [Dmt1]DALDA internalization. [Dmt1]DALDA uptake was also observed in SH-SY5Y, human embryonic kidney 293, and CRFK cells, and was independent of whether the cells expressed opioid receptors. The efflux of [3H][Dmt1]DALDA from Caco-2 cells was temperature-dependent and was inhibited by DEPC, but was not affected by verapamil, an inhibitor of P-glycoprotein. These data show transcellular translocation of a highly polar 3+ charge tetrapeptide and suggest that [Dmt1]DALDA may not only distribute across the blood-brain barrier but also it may even have reasonable oral absorption.

A highly potent peptide analgesic that protects against ischemia-reperfusion-induced myocardial stunning.

We recently discovered an opioid peptide analgesic, 2',6'-dimethyltyrosine (Dmt)-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA), that can protect against ischemia-induced myocardial stunning. In buffer-perfused hearts, 30-min global ischemia followed by reperfusion resulted in a significant increase in norepinephrine (NE) overflow immediately upon reperfusion and significant decline in contractile force (45%). Pretreatment with [Dmt(1)]DALDA before ischemia completely abolished myocardial stunning and significantly reduced NE overflow (68%). In contrast, pretreatment with morphine before ischemia only provided brief protection against myocardial stunning and no reduction in NE overflow. [Dmt(1)]DALDA inhibited [(3)H]NE uptake into cardiac synaptosomes in vitro (IC(50) = 3.9 microM), whereas morphine had no effect. Surprisingly, protection against myocardial stunning was apparent even when hearts were perfused with [Dmt(1)]DALDA only upon reperfusion, whereas reperfusion with morphine had no effect. Binding studies with [(3)H][Dmt(1)]DALDA revealed no high-affinity specific binding in cardiac membranes, suggesting that the cardioprotective actions of [Dmt(1)]DALDA are not mediated via opioid receptors. These findings suggest that [Dmt(1)]DALDA is a potent analgesic that may be useful for myocardial stunning resulting from cardiac interventions or myocardial ischemia.

Profound spinal tolerance after repeated exposure to a highly selective mu-opioid peptide agonist: role of delta-opioid receptors.

Recent studies suggest that delta-opioid receptors play a role in the development of opioid tolerance and led us to hypothesize that highly selective mu-opioid agonists may produce less tolerance. H-2',6'-dimethyltyrosine-D-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA) has extraordinary selectivity for mu-receptors (K(i)(delta)/K(i)(mu) > 14,000). Daily administration of [Dmt(1)]DALDA (5 times ED(50); s.c.) for 7 days increased ED(50) 3.6-fold from 0.16 to 0.58 micromol/kg. A higher dose of [Dmt(1)]DALDA (10 times ED(50), every 12 h) for 2.5 days resulted in a 11.7 times increase in the ED(50) (1.9 micromol/kg). Complete cross-tolerance to morphine was observed, with a 3.4- and 15.1-fold shift in the morphine ED(50), respectively. We also compared the extent of spinal versus supraspinal tolerance after repeated s.c. [Dmt(1)]DALDA administration. Five doses of [Dmt(1)]DALDA (10 times ED(50), every 12 h) resulted in a 3.4 times shift in the i.c.v. ED(50) (15.4 versus 4.6 pmol/mouse) but a 44 times shift in the i.t. ED(50) (52.9 versus 1.2 pmol/mouse). Tolerance to [Dmt(1)]DALDA was associated with 30 to 35% reduction in [(3)H][Dmt(1)]DALDA binding in brain and spinal cord. Coadministration of [Dmt(1)]DALDA with delta-antagonist naltriben (NTB) reduced spinal tolerance by 50%. Even after spinal tolerance had been established, addition of a delta-antagonist (NTB or H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH) significantly enhanced the potency of i.t. [Dmt(1)]DALDA 2- to 4-fold. These results suggest that agonist activation of delta-receptors is not necessary for the development of opioid tolerance; however, delta-receptors play a modulatory role in the maintenance of the tolerant state.