Robert C.A. Frederickson

Enkephalin analogues and naloxone modulate the release of growth hormone and prolactin--evidence for regulation by an endogenous opioid peptide in brain.

Abstract Met 5 -enkephalin amide, D-Ala 2 -Met 5 -enkephalin amide, D-Ala 2 -Leu 5 -enkephalin amide, morphine sulfate and naloxone hydrochloride were examined for their effects on growth hormone and prolactin release in vivo and in vitro . Subcutaneous injection of D-Ala 2 -Met 5 enkephalin amide a , D-Ala 2 -Leu 5 enkephalin amide b and morphine sulfate, but not Met 5 -enkephalin and amide c , resulted in significant elevations in the serum growth hormone and prolactin of immature female rats. Naloxone blocked the hormone-stimulatory effect of the opioid receptor agonists and when administered alone significantly reduced serum growth hormone and prolactin concentrations. None of the drugs demonstrated a direct action on anterior pituitary tissue growth hormone or prolactin release in vitro .

A fragment of substance P with specific central activity: SP(1–7)

Amino-terminal fragments of substance P (SP), SP(1-7) and SP(1-8), were found to produce naloxone-reversible antinociception in the mouse similar to that produced by SP. Similar to SP, these peptides produce antinociception only within a narrow dose range. They have no activity on smooth muscle or blood pressure. These results suggest that contrary to peripheral effects of SP, which are mediated by receptors which recognize the carboxy-terminal part of the SP molecule, certain central actions of SP are mediated by receptors which recognize the amino-terminal part of the SP molecule. SP may be metabolized to this active fragment prior to its action at these receptors.

Diurnal differences in opioid peptide levels correlated with nociceptive sensitivity

Abstract An increase in whole mouse brain total opioid levels, determined by mouse was deferens bioassay, was observed in mice sacrified in late afternoon (when they are least responsive to pain) compared to early morning (when they are most responsive to pain). There were no comparable increases in levels of met 5 - or leu 5 - enkephalin measured by RIA methodology. There were differences, however, in the effects of noxious stimuli on met 5 -enkephalin levels in the efternoon compared to the morning. In the afternoon, but not in the morning, the levels of met 5 -enkephalin in mice tested after hot plate stress were significantly increased compared to those of unstressed animals. Thus, there appears to be some correlation between activity in endogenous opioid systems and the ability of mice to withstand noxious stimuli.

K-stimulated release of leu- and met-enkephalin from rat striatal slices: Lack of effect of morphine and naloxone

The release of leu- and met-enkephalin from rat striatal slices was determined by superfusing the slices in vitro and running the superfusates directly over columns of Amberlite XAD-2 from which the peptides were eluted with methanol and measured by radioimmunoassay. Depolarization by high K concentrations increased the release of both pentapeptides many fold; the degree of increase, however, depended in part on the length of time chosen for the stimulation period, suggesting that the stimulation effect was very short lived. The stimulated release of both peptides (but not the resting release) was at least partially dependent on Ca in the medium and was totally inhibited by high Mg concentrations. Selected concentrations of naloxone and morphine in the superfusing medium had no effect on the resting or stimulated release of the peptides. The results support the hypothesis that these peptides serve as neurotransmitters in the striatum, but autoregulation of their release by morphine and naloxone could not be demonstrated.

The role of the pituitary in the diurnal variation in tolerance to painful stimuli and brain enkephalin levels

Abstract Studies were carried out on hypophysectomized rats and mice in comparison to sham-operated controls in order to assess the role of the pituitary in the diurnal rhythm in sensitivity to pain, the hyperalgesic effect of naloxone and the effect of stress on brain levels of met-enkephalin. There were no significant differences in jump latencies between hypophysectomized and sham-operated control mice. The jump latencies in the p.m. were significantly greater than those in the a.m. for both the sham and the hypophysectomized mice. In both the sham and hypophysectomized mice and rats, naloxone significantly reduced the jump latencies in the p.m. The stress-induced increase in the p.m. of brain met-enkephalin, furthermore, persisted in the hypophysectomized rats. We conclude that the pituitary is not essential for the diurnal variation in responsivity to pain, the hyperalgesic activity of naloxone or the stress-induced increases in brain met-enkephalin.

Metkephamid (Tyr-D-ala-Gly-Phe-N(Me)Met-NH2), a potent opioid peptide: receptor binding and analgesic properties.

The interaction of metkephamid (Tyr-D-Ala-Gly-Phe-N(Me)Met-NH2) with 3H-dihydromorphine and 3H-D-Ala2-D-Leu5-enkephalin binding has been examined in rat brain homogenates. Displacements of both 3H-ligands by metkephamid indicate that metkephamid interacts competitively with greatest potency to the high affinity binding component for both ligands (mu1 site). Unlike most enkephalins and opiates, metkephamid binds equipotently to both morphine-selective (mu2) and enkephalin-selective (delta) binding sites. Metkephamid is differentiated from morphine by its better than 12-fold higher affinity for the delta receptor. Blockade of the high affinity (mu1) binding in vivo with high doses of naloxazone dramatically reduces metkephamids analgesic potency.

Effects of naloxone and acetylcholine on medial thalamic and cortical units in naive and morphine dependent rats

Abstract An important role has been suggested for acetylcholine (ACh) in both the development and the expression of dependence upon morphine (1). However, the specific nature of this role has not been established. Much recent evidence implicates the medial thalamic (m. thalamic) region in the genesis of these phenomena. In particular, Wei et al. (2) report this area to be one of the most effective brain regions for the precipitation of withdrawal (WD) in opiate dependent rats by the direct implantation of naloxone. Furthermore, electrical stimulation of the m. thalamic region of naive rats can produce a series of responses reminiscent of the morphine WD syndrome (unpublished observations). Thus naloxone may precipitate WD in opiate dependent animals by exciting neurons in the m. thalamic region which are not similarly excited in naive animals. It might do this by releasing increased stores of ACh onto receptors made supersensitive secondary to inhibition of ACh release by morphine (2,3,4). However, recent work suggests that such a simplistic mechanism is probably inadequate to explain opiate WD (1). The present experiments were performed to test this theory more directly using the technique of microiontophoresis. The results indicate that the number of neurons excited by naloxone is increased in m. thalamus, but not cerebral cortex, of dependent animals. However, this cannot be attributed simply to a release of ACh by naloxone and receptors appear subsensitive rather than supersensitive to ACh in dependent animals.

Enantiomers of [R,S]-thiorphan: Dissociation of analgesia from enkephalinase a inhibition

The [R] and [S] enantiomers of the enkephalinase A inhibitor [R,S]-thiorphan have been prepared by asymmetric synthesis. The [S] isomer is principally responsible for the angiotensin converting enzyme inhibitory activity of [R,S]-thiorphan, whereas there were only small differences in the ability of the [R] and [S] isomers to inhibit enkephalinase both in vivo and in vitro. In contrast, the in vivo analgesic activity of [R,S]-thiorphan resided principally in the [R] isomer. These data indicate a surprising dissociation of enkephalinase inhibition from analgesic activity. The fact that the two enantiomers of [R,S]-thiorphan were effective inhibitors of enkephalinase, yet the [R] isomer had substantially greater analgesic activity, indicates that factors other than enkephalinase inhibition may be important for [R, S]-thiorphans analgesic properties.

Cross-tolerance studies distinguish morphine- and metkephamid-induced analgesia.

In vitro data demonstrate that metkephamid (LY127623), an analog of methionine enkephalin, has a high affinity for the delta opioid receptor, as well as the mu-receptor. Data generated utilizing two in vivo measures of receptor selectivity, furthermore, indicate that metkephamids analgesic activity is in part mediated by delta opioid receptors. The analgesic activity of metkephamid was investigated in the mouse writhing assay following chronic treatment with morphine, the prototypic mu agonist. Mice were treated chronically with increasing doses of morphine or saline and the inhibition of writhing measured in response to an acute injection of morphine or metkephamid. The dose response curve for morphine was shifted to the right 3- to 4-fold following chronic administration of morphine. In contrast, no such shift in the dose response curve for metkephamid was observed in these morphine-tolerant mice. In a further series of tests, a 50 mg/kg dose of naloxazone 20 hr prior to the assessment of morphine or metkephamid analgesia in the mouse hot plate test substantially shifted the dose-response curve for morphine to the right, while leaving the dose-response curve for metkephamid unchanged. These results suggest that delta-receptor activation contributes to the analgesia produced by metkephamid.

Human endorphin: Comparison with porcine endorphin, enkephalin and normorphine

Abstract We have extracted a material from human brain obtained at autopsy which has morphine-like activity on the mouse vas deferens preparation. This human endorphin behaved similarly on Sephadex G-15 and cation exchange columns as did porcine material and there were no qualitative differences in pharmacological activity between this material and porcine endorphin or enkephalin. Further purification of the human material is in progress. These results add further support to the suggestion that the stereospecific binding sites in opiate responsive species are receptors for an endogenous peptide ligand with a role in brain function.