Comparative effects of Pro-Leu-Gly-NH2 and cyclo(Leu-Gly) administered orally on the development of tolerance to the analgesic effect of morphine in the rat

Comparative effects of Pro-Leu-Gly-NH2 (MIF) and cyclo(Leu-Gly) (CLG) administered orally at different stages of chronic morphine treatment on the development of tolerance to the analgesic effect of morphine in the rat were determined. Male Sprague-Dawley rats were implanted with either 6 placebo or morphine pellets during a 7-day period. Implantation of morphine pellets resulted in the development of a high degree of tolerance as evidenced by a decrease in the analgesic response to morphine. Administration of CLG (8 and 16 mg/kg/day) on day 5, 6 and 7 of implantation inhibited the development of tolerance to morphine but 4 and 32 mg/kg doses had no effect. Further, CLG (2 mg/kg/day for 7 days) inhibited the development of tolerance but higher doses (4 and 8 mg/kg) had no effect. MIF (26 and 52 mg/kg) administered orally on the last three days of the implantation schedule inhibited the development of tolerance to morphine. MIF (6.5 mg/kg/day for 7 days) inhibited the development of tolerance but the higher doses had no effect. Concurrent administration of MIF (6.5 mg/kg) and CLG (2 mg/kg) for seven days failed to inhibit the development of tolerance. A single dose of MIF or CLG administered a day before the assessment of tolerance did not affect the morphine tolerance. Thus, even after a significant degree of tolerance to morphine had developed, neuropeptides like MIF and CLG given orally, in appropriate doses, can inhibit development of tolerance to morphine and restore the analgesic effect of morphine.     

The effect of oxytocin and fragment (MIF-I) on the development of tolerance to hypothermic and hypnotic action of ethanol in the rat

The daily pretreatment of rats with oxytocin (OXY) or MIF-I prior to ethanol (Et-OH) administration markedly altered the alcohol tolerance when tested on the fifth day of treatment. OXY (800 and 2400 nmole/kg SC) and MIF (800 nmole/kg SC) inhibited the development of tolerance to the hypnotic effect of Et-OH. MIF at this dose also inhibited the tolerance to the hypothermic effect. Only OXY in the dose of 800 nmole/kg suppressed hypothermia in an acute experiment with Et-OH and produced by itself hypothermia after acute administration (2400 nmole/kg). The tolerance to this last effect developed after four days of peptide treatment. The results indicate that OXY and MIF-I can influence the processes of development of tolerance to some central depressive effects of Et-OH in rats.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Substance P

Substance P is a potent hypotensive peptide first noted in extracts of equine brain and intestinal tissue in 1931 that also stimulates the contraction of various intestinal tissues. Its isolation proved to be particularly difficult and was not accomplished until 1970 following the chance finding of a sialogogic factor in bovine hypothalamic extracts. Utilizing this biological property to monitor purification procedures, the active sialogogic peptide was obtained in pure form and identified as Substance P by multiple chemical and biological criteria. The amino acid sequence of Substance P is HArgProLysProGlnGlnPhePheGlyLeuMetNH₂. Synthetic Substance P has been prepared and a radioimmunoassay has been developed, thus facilitating well-defined research into its physiological roles. Much of the current research is centering on its possible role in sensory neurotransmission.     

Pharmacological activities of the MIF-1 analogues Pro-Leu-Gly, Tyr-Pro-Leu-Gly and pareptide

The pharmacological activities of the related free acid analogues of MIF-1, Pro-Leu-Gly (PLG) and Tyr-Pro-Leu-Gly (YPLG), were investigated because of the possibility that they may be formed during the digestion of milk and wheat proteins in vivo. The amino acid sequences-Tyr-Pro-Leu-Gly- and -Pro-Leu-Gly- are present in proteins from these foods. Chronic administration of either PLG (0.25 mg/kg, SC, BID) or the control substance, pareptide (0.25 mg/kg, SC, BID), antagonized the development of tolerance to the cataleptic effects of haloperidol in mice. The effect of YPLG (0.25 mg/kg, SC, BID) on the development of this tolerance was borderline and not statistically significant. Nanomolar concentrations of PLG, YPLG, and pareptide each increased the in vitro binding of ³H-apomorphine to rat striatal receptors. In this in vitro system, bell-shaped dose response curves were observed for each peptide. The effects of these peptides on tolerance development and apomorphine binding are similar to those previously reported for MIF-1 and demonstrate that amidation at the carboxyl terminus is not required for biological activity.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Neuropharmacological activity of cerucal and haloperidol by intraperitoneal and intrastriatal administration in rats]

The effects of three-week administration of cerucal and haloperidol were compared in two ways of injections--intraperitoneal 10 mg/kg and intrastriatal 5 micrograms/l microliter. Both of the ways produce the inhibition of rats locomotor activity, but only after the intraperitoneal haloperidol injection the cataleptic status was observed. The inhibition of avoidance conditioning in shuttle box was registered in each haloperidol microinjections, but in case of cerucal intrastriatal microinjection this effect in first 4-5 days of experiment was limited. No any muscular deviation after cerucal injection (systemic and intrabrain) were observed. The antihyperkinetic activity and striatal mechanisms of cerucal and their application in neurology were discussed.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Pro-Leu-Gly-NH2 and pareptide inhibit development of tolerance to haloperidol catalepsy in the mouse

Single doses of MIF-1 (0.03-2.0 mg/kg, SC) and chronic pretreatments with MIF-1 (0.03-2.0 mg/kg, SC, BID, 3 1/2 days) or pareptide (0.25 mg/kg, SC, BID, 3 1/2 days) did not affect the acute cataleptic response to haloperidol in the mouse. Chronic pretreatment with haloperidol (8.0 mg/kg, IP, BID, 3 days) decreased the duration of catalepsy in mice given smaller challenge dose of haloperidol (2.0 or 3.0 mg/kg, IP) 15 hours after the last pretreatment injections. Administration of either MIF-1 or pareptide to mice also chronically pretreated with haloperidol antagonized the development of tolerance.     

Some behavioural effects of carbamazepine - comparison with haloperidol.

The experiments presented in this paper aimed to investigate the influence of atypical antiepileptic drug carbamazepine (CBZ, CAS 298-46-4) classified also as normothymic drug on spatial memory in Morris water maze test and anxiolytic effect in two-compartment exploratory test in rats. The study also investigated the probably occurring side effects (measuring cataleptic activity and motor coordination) following single and chronic administration of CBZ compared to haloperidol (HAL, CAS 52-86-8), a conventional antipsychotic. All the tests were carried out on male Wistar rats. CBZ 30 mg/kg was administered orally 60 min before the tests and HAL 0.15 mg/kg was administered orally 60 min before the tests. In the Morris test memory improvement only after chronic administration of CBZ on the 7 and 14 day of treatment was observed, whereas after 14 days of HAL treatment spatial memory impairment was noted. In the two-compartment exploratory test 30 mg/kg of CBZ had an anxiolytic effect after 7 and 14 days of treatment, whereas HAL did not show anxiolytic effect after single and chronic treatment. CBZ did not induce catalepsy after single as well as chronic administration. HAL evoked a strong cataleptic effect both after acute and chronic treatment. CBZ had no impact on motor coordination in the chimney test and HAL disturbed motor coordination in rats after single as well as chronic administration. CBZ may be an useful normothymic drug using in bipolar affective disorder treatment with co-occurred anxiety and cognitive deficits. The lack of significant side effects of CBZ may be an alternative way of treatment in comparison with older drugs, such as lithium carbonate.     

Dependence of the effects of tripeptide MIF and lithium chloride on the degree of supersensitivity of dopamine receptors of the brain

The desensitizing effects of MIF and lithium in respect to supersensitive striatal DA receptors in rats with unilateral lesion of the nigro-striatal pathway by 6-hydroxydopamine were revealed. Two groups of 6-hydroxydopamine-denervated rats were selected by their qualitative responsiveness to apomorphine-induced rotational behavior. It was found, that MIF and lithium (subchronic administration) did not modify behavioral supersensitivity in the highly sensitive group which showed two-peak rotational pattern in response to 0.05 mg/kg apomorphine, which was converted into a single-peak rotational pattern by haloperidol. On the contrary, administration of MIF and lithium for 21 days inhibited the apomorphine-induced rotations to 54 and 65% respectively in the less supersensitive group, which showed a single peak rotational pattern to apomorphine. Moreover, haloperidol showed the high antagonistic potency in these animals. These results suggest, that MIF and lithium might not exert desensitizing effects in the presence of high degree of supersensitivity of the striatal DA receptors, which is probably involved, for example, in the phenomenon of persistent tardive dyskinesia.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Attenuation of morphine tolerance and dependence by alpha-melanocyte stimulating hormone(alpha-MSH).

Repeated administration of morphine resulted in significant reduction of its analgesic potency. If 0.1 mg/kg α-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance. Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma α-MSH level at the time of morphine administration. Of the morphine abstinence symptoms the naloxone-induced jumping in morphine pretreated mice could not be modified either by α-MSH coadministration or by MIF pretreament, but the withdrawal body weight loss was found to be diminished by the former and increased by the latter peptide. The possible role of α-MSH in preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed.     

Role of N-methyl-D-aspartate (NMDA) receptors in experimental catalepsy in rats

N-methyl-D-aspartic acid (NMDA; 40 mg/kg, i.p.) did not elicit catalepsy, but it potentiated the cataleptic effect of haloperidol and GABAB receptor agonist, baclofen. MK-801 (0.2 mg/kg, i.p.), NMDA-receptor antagonist, reversed haloperidol- but not baclofen-induced catalepsy. MK-801 also potentiated the anticataleptic effect of scopolamine and bromocriptine against haloperidol-induced catalepsy. Dihydropyridine (DHP) calcium-channel antagonists such as nimodipine and nitrendipine (10 mg/kg, i.p.), reversed the anticataleptic effect of MK-801, and potentiated the cataleptic effect of haloperidol, as well as baclofen. These observations indicate the involvement of NMDA receptors in catalepsy, and suggest a potential clinical implication of NMDA-receptor antagonists in Parkinson's disease.     

Isolation and structure identification of a morphine-like peptide "enkephalin" in bovine brain.

The ability of bovine brain extracts to compete in a selective fashion for opiate receptor binding is attributable to a small peptide. The substance has been purified to homogeneity and identified as comprising two penta-peptides HTyrGlyGlyPheLeuOH (Leucine-enkephalin) and HTyrGlyGlyPheMetOH (methionine enkephalin). Bovine brain contains 4 times as much leucine-enkephalin as methionine-enkephalin in contrast to pig brain in which these ratios are reversed. Competition for opiate receptor binding by leucine-enkephalin is reduced more by sodium and enhanced more by manganese than is the case for methionine-enkephalin, suggesting that leucine-enkephalin may be a “purer” agonist than methionine-enkephalin.     

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Similarities between the akinesia induced by carbachol microinjections into the pontine reticular formation and neuroleptic catalepsy.

We reported previously that microinjections of carbachol directly into the pontine reticular formation of rats induced intense akinesia. In the present article we report results of tests for rigidity, righting, bracing and clinging which were conducted with the purpose to characterize behaviorally this type of akinesia. After injections of 5-15 micrograms/0.5 microliter of carbachol into the pontine reticular formation the rats were cataleptic, were not rigid when equilibrium was not challenged, had strong righting reflexes and strong bracing and clinging responses. This type of akinesia is different from the catatonia induced by systemic morphine (20 mg/kg IP), but similar to the catalepsy induced by systemic injections of haloperidol (5 mg/kg IP). It is thus suggested that the cataleptic state produced by topical carbachol in the pons is related to the dopaminergic mechanisms important for the cataleptic effect of the neuroleptic drugs.     

Rabbit liver fructose 1,6-bisphosphatase: labeling of the active and allosteric sites with pyridoxal 5-phosphate and sequence of a nonapeptide from the active site

The active and allosteric sites of fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) were labeled by reaction with pyridoxal phosphate and sodium borohydride in the presence of the allosteric inhibitor AMP or the substrate, Fru-P₂, respectively. Modification of the active site results in loss of activity. Modification of the allosteric site decreases the sensitivity of the enzyme to inhibition by AMP and alters its ability to bind to blue dextran-Sepharose. The allosteric and active sites have been located on different cyanogen bromide peptides; the sequence of a nonapeptide from the active site is (H)GlyLysLeuArgLeuLeu TyrGluCys(OH). The lysyl residue is modified by pyridoxal phosphate.