Studies on the mechanism of thyrotropin releasing hormone induced inhibition of gastrointestinal transit

Intracerebral administration of thyrotropin releasing hormone (TRH) inhibited gastrointestinal transit in the mouse as determined by the charcoal meal test. A similar inhibitory effect was produced by morphine administered subcutaneously. TRH enhanced morphine-induced inhibition of gastrointestinal transit. Intracerebral injections of cyclo (His-Pro), a postulated metabolite, did not affect gastrointestinal transit either by itself or that produced by morphine. It is suggested that gastrointestinal transit effects of TRH are not mediated via its conversion to cyclo (His-Pro).     

The role of T-type calcium channels in morphine analgesia,development of antinociceptive tolerance and dependence to morphine,and morphine abstinence syndrome

Involvement of T-type voltage dependent Ca2+ channels (VDCCs) on morphine antinociception, in the development of tolerance and dependence to morphine, and naloxone-precipitated abstinence syndrome in morphine dependent mice was examined by using mibefradil, a T-type VDCCs blocker. Mice were rendered tolerant and dependent on morphine by subcutaneous (s.c.) implantation of a morphine pellet containing 75 mg of morphine base for 72 hr. The tail-flick test was used to assess the nociceptive threshold. Coadministration of acute mibefradil (10 mg/kg, i.p.) with morphine enhanced the antinociceptive effects of acute morphine. Repeated mibefradil administration (10 mg/kg, i.p., just before, 24 and 48 hr after morphine pellet implantation) completely blocked the development of tolerance to the antinociceptive effect of morphine and even by this effect reached supersensitivity to morphine. However, repeated mibefradil treatment did not alter the development of dependence to morphine assessed by the A(50) values of naloxone (s.c.) required to precipitate withdrawal jumping 72 hr after morphine pellet. But, acute mibefradil (10, 30, and 50 mg/kg, i.p.) dose dependently decreased the expression of morphine abstinence syndrome when given directly 30 min prior to naloxone (0,05 mg/kg, s.c.) 72 hr after morphine pellet. These results indicate a critical role of T-type VDCCs in morphine antinociception, the development of tolerance to the antinociceptive effects of morphine and in morphine abstinence syndrome.     

Brain levels of morphine in mice following removal of a morphine pellet and naloxone challenge: no evidence for displacement

Male ICR mice were rendered tolerant to and dependent on morphine by subcutaneous implantation of a 75 mg morphine pellet for 72 hours. At 2, 4, and 6 hours after pellet removal groups of 7–10 mice were challenged with ip saline or naloxone and their brain concentrations of morphine estimated by radioimmunoassay (RIA). The brains were prepared for RIA by either organic or inorganic (0.01 N HC1) extraction and in most experiments the two methods were shown to be equivalent with respect to the final concentration of morphine. There was no difference in brain morphine between saline and naloxone (10 mg/kg) treated groups when they were challenged 4 hours after pellet removal and sacrificed 1, 5, 10, 15, 20, 30, 45, and 60 minutes later. In contrast, when the challenge was administered 6 hours after pellet removal the naloxone treated groups has higher concentrations of brain morphine than the saline controls. Brain levels in mice that received 0.10, 1.0, 10, 100 mg/kg naloxone did not differ consistently from saline controls. We found no consistent evidence that naloxone decreases the concentration of morphine in brain homogenates obtained from mice during the initial 6 hours after pellet removal.     

Structure activity relationship studies with hypothalamic peptide hormones. II. Effects of thyrotropin releasing hormone analogs on morphine-induced responses in mice

The effects of several analogs of thyroliberin (TRH), that have a chloro-acetyl substituent at the amino terminus, on locomotor depressant, locomotor stimulant, hyperthermic and hypothermic response to morphine were determined in the mouse. These compounds included N-(chloroacetyl)-L-phenylalanylpyrrolidine (ClAc-Phe-Pyrr), N-[m-(chloroacetyl)benzoyl]-L-phenylalanylpyrrolidine] (mClAcBz-Phe-Pyrr), N-[m-(chloroacetyl)benzoyl]-L-alanyl-L-phenylalanylpyrrolidine (mClAcBz-Ala-Phe-Pyrr), N-[p-(chloroacetyl)benzoyl]-L-alanyl-L-phenylalanyl-pyrrolidine (pClAcBz-Ala-Phe-Pyrr), N-(chloroacytyl)-L-alanyl-L-phenylalanyl-L-prolineamide(ClAc-Ala-Phe-Pro-NH₂), N-[m-(chloroacetyl)-benzoyl]-L-phenylalanyl-L-prolineamide (mClAcBz-Phe-Pro-NH₂), N-[p-(chloroacetyl)benzoyl]-L-phenylalanyl-L-prolineamide (pClAcBz-Phe-Pro-NH₂). Since TRH is metabolized to cyclo (His-Pro) and the latter is shown to possess TRH like activity, an analog cyclo (Phe-Pro) was also used. Administration of morphine to mice at 10 mg/kg ip produced hyperthermia and depression in locomotor activity, while at 80 mg/kg ip, hypothermia and stimulation in locomotor activity were observed. Intracerebral injection of the following peptides (10 μg each per mouse) administered 10 min prior to morphine injection antagonized locomotor depression, hyperthermia, locomotor stimulation and hypothermia induced by an appropriate dose of morphine: mClAcBz-Phe-Pyrr, pClAcBz-Ala-Phe-Pyrr, ClAcAla-Phe-Pro-NH₂, pClAcBz-Phe-Pro-NH₂, cyclo (Phe-Pro) and TRH. The compounds which had no effect on low dose or high dose morphine induced responses included pGlu-Phe-Pyrr, mClAcBz-Ala-Phe-Pyrr, and mClAcBz-Phe-Pro-NH₂. One compound, namely ClAc-Phe-Pyrr, antagonized morphine-induced locomotor stimulation and hypothermia but did not affect locomotor depression and hyperthermia produced by morphine. None of these peptides had any effect on the body temperature or the locomotor activity of normal mice. Many of the active compounds were previously shown to possess extremely weak or no activity in releasing thyrotropin from the pituitary. It is concluded that several of these analogs of TRH possess CNS activity in antagonizing morphine effects, and that a lack of relationship exists between the CNS and endocrine activity of these peptides.     

Is all cyclo(His-Pro) derived from thyrotropin-releasing hormone?

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is an endogenous cyclic dipeptide that is ubiquitously distributed in tissues and body fluids of both man and animals. This cyclic dipeptide is not only structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-ProNH₂), but it can also arise from TRH by the action of the enzyme pyroglutamate amino-peptidase (pGlu-peptidase). The data on the distribution of TRH, cyclo(His-Pro), and pGlu-peptidase under normal and abnormal conditions are summarized and potential relationships analyzed. We conclude that all of the cyclo(His-Pro) cannot be derived from TRH. Two additional sources of cyclo(His-Pro) are suggested. It is proposed that 29,247 molecular weight TRH prohormone, prepro TRH, which contains 5 copies of TRH sequence, can be processed to yield cyclo(His-Pro). Thus, both TRH and cyclo(His-Pro) share a common precursor, prepro[TRH/Cyclo(His-Pro)].     

Unresponsiveness of GH cells to cyclo(histidyl-proline), a metabolite of thyrotropin releasing hormone

Cyclo(Histidyl-Proline) is a metabolite of thyrotropin-releasing hormone. It has been suggested that this peptide plays a role in regulating prolactin secretion in GH cells. An investigation of the effect of cyclo(His-Pro) on GH cells indicated that it does not affect basal prolactin release or accumulation or the levels stimulated by TRH. cAMP levels in GH cells are elevated by TRH or VIP, but not influenced by cyclo(His-Pro). cGMP levels in GH cells are not affected by either TRH or cyclo(His-Pro). While there is specific binding of TRH to receptors in GH cells, no such receptors for cyclo(His-Pro) are detectable. It is suggested that GH cells are unresponsive to cyclo(His-Pro).     

The effect of thyrotropin releasing hormone and morphine on gastrointestinal transit

The effect of thyrotropin releasing hormone (TRH) alone and in combination with morphine on the gastrointestinal transit was investigated by using the charcoal meal test in mice. The intraperitoneal (IP) administration of TRH decreased the transit when given in a dose of 1.0 mg/kg 10 min prior to the meal. The intracerebroventricular (ICV) administration of TRH (10 μg/mouse) also inhibited the transit when given just prior to the charcoal meal. Subcutaneous (SC) administration of morphine (5, 10 and 20 mg/kg) inhibited gastrointestinal transit in a dose dependent manner. When TRH (1, 3 and 10 mg/kg, IP as well as 0.3 μg, ICV) which had no effect on the transit by itself was combined with morphine (10 mg/kg, SC), an enhancement in the inhibition of the transit was observed. TRH-induced inhibition of the transit was antagonized by naloxone (0.1 mg/kg, SC). It is concluded that TRH inhibits gastrointestinal transit in the mouse possibly via the opiate receptor system.     

Histidyl-proline diketopiperazine: its biological role as a regulatory peptide

Summary Histidyl-proline diketopiperazine [cyclo(His-Pro)] is a metabolic of thyrotropin releasing hormone (TRH). This review summarizes the literature concerning cyclo (His-Pro) and, in addition, some studies dealing with TRH and other peptides that are considered of interest. The enzymes concerned with the metabolism of TRH are discussed. Distribution studies of peptides by immunological methods show that, while TRH is concentrated in synaptosomes, cyclo (His-Pro) is not, suggesting that cyclo (His-Pro) is not a classical neurotransmitter. Rat brain contains approximately three times as much cyclo (His-Pro) as TRH, mainly localized in the pituitary and hypothalamus. While the TRH is found in a free form, the cyclo (His-Pro) is bound to a carrier of molecular weight approximately 70 000. While specific membrane receptors for TRH have been detected in pituitary cells, no such receptors for cyclo (His-Pro) have yet been found in brain or pituitary; however, there is a specific binding of cyclo (His-Pro) to adrenal cortex membranes, Both TRH and cyclo (His-Pro) have effects in the central nervous system or pituitary. These include effects on prolactin release, thermoregulation, CNS depression, stereotypic behavior and cyclic nucleotide levels. Possible mechanisms and interrelations of these effects are discussed.     

Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor,NG-nitro-L-arginine methyl ester

The hypothesis that an arginine-nitric oxide (NO) synthase-NO system mediates the morphine abstinence syndrome was tested in adult male rats implanted subcutaneosly for 3 days with one morphine (75 mg) pellet followed by naloxone-precipitated withdrawal (0.5 mg/kg). Injection with a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (NAME, 100 mg/kg subcutaneous), shortly before naloxone-induced withdrawal significantly inhibited abstinence signs by 25–80%. Continuous infusion of NAME via subcutaneous osmotic pumps during the development of morphine physical dependence and during naloxone-precipitated withdrawal also inhibited morphine abstinence signs. In addition, treatment with isosorbide dinitrate, a NO donor, induced a quasi morphine-abstinence syndrome (QMAS) that was significantly suppressed by implantation of a morphine pellet 3 days before isosorbide dinitrate treatment. These results indicate that NO mediates part of the expression of the morphine abstinence syndrome.     

Lack of effect of histidyl-proline diketopiperazine on basal level and TRH-induced response of cytosolic calcium concentration in rat lactotrophs.

Histidyl-proline diketopiperazine [cyclo(His-Pro)] has recently been shown to inhibit prolactin (PRL) secretion in vitro and in vivo. This peptide is well known as a metabolite of thyrotropin-releasing hormone (TRH), which is one of the endogenous secretagogues of PRL. In this study, we investigated the effect of cyclo (His-Pro) on the cytosolic Ca2+ concentration [[Ca2+]i) in cultured lactotrophs by using a lactotroph-enriched fraction separated from female rat pituicytes by centrifugal elutriation. TRH (10 nM) induced a rapid rise in [Ca2+]i in the lactotrophs, followed by a plateau phase of prolonged increase in [Ca2+]i. In contrast, the addition of 100 microM of cyclo (His-Pro) caused no changes in the basal level or the TRH-induced plateau response of [Ca2+]i. Although pretreatment with cyclo (His-Pro) tended to decrease the biphasic increase in [Ca2+]i induced by TRH, the inhibitory effect was not statistically significant. These results demonstrated that cyclo (His-Pro) has no effect on [Ca2+]i in lactotrophs, and does not affect the TRH-induced increase in [Ca2+]i, indicating that the inhibition of PRL secretion by cyclo (His-Pro) may be primarily mediated by other intracellular messengers such as cyclic nucleotides and secondarily involved in other inhibitory systems including that of dopamine.     

Differentiation of beta-adrenoceptors in right atrium, diaphragm and adipose tissue of the rat, using stereoisomers of propranolol, alprenolol, nifenalol and practolol.

2-Diazomorphine-bovine serum albumin (2-DAM-BSA) was prepared by diazotizing p-aminobenzoyl-BSA to morphine. Rabbits immunized with 2-DAM-BSA produced antibodies directed to morphine. A 50 percent reduction in ³H-morphine binding required 4.4 pmol of morphine, and 60, 225, and 350 pmol of normorphine, morphine-3-glucuronide, and codeine, respectively. A radioimmunoassay for brain morphine is described, validated, and used to determine if naloxone alters brain morphine in morphine pelleted mice. The apparent biological half-life of morphine in brain was approximately 52 hours between 24 and 72 hours after pellet implantation, and decreased to 1.25 hours after pellet removal. Naloxone (10 mg/kg) administered 24, 48, or 72 hours after implantation and in doses of 1.0–100 mg/kg administered at 48 hours resulted in either no significant change, or, in a few experiments, increased the brain concentration of morphine. The present experiments could not detect a fraction of total brain morphine that is reduced by naloxone.     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with $\text{pyroglutamate aminopeptidase}$ activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.     

On the mechanism of fasting-associated elevations in hypothalamic cyclo(His-Pro) content

Potential mechanism(s) underlying the fasting-associated rise in hypothalamic cyclo(His-Pro) content was explored by examining the effects of 24-hour fasting on: (i) cyclo(His-Pro) synthesis from TRH, (ii) cyclo(His-Pro) metabolism, and (iii) cyclo (His-Pro) secretion by hypothalamic tissue in vitro. The data presented here show that none of these three variables were altered due to fasting. Two additional potential changes that could cause cyclo(His-Pro) elevations during fasting are suggested. These include an in vivo decrease in hypothalamic cyclo(His-Pro) secretion that may not be apparent in vitro, and/or an increase in the synthesis of cyclo(His-Pro) from a precursor(s) other than TRH.     

[3H]cyclo(Histidyl-Proline) in rat tissues: Distribution,clearance and binding

Cyclo(Histidyl-Proline), a metabolite of TRH, has been demonstrated to have a number of biological activities. The clearance, distribution and binding of the peptide in the rat was studied. Cyclo(His-Pro) was cleared from the circulation biphasically ($\text{tl}\text{2}\text{= 1.25 and 33 min}$). Unmetabolized cyclo(His-Pro) appeared rapidly in urine. Accumulation of [³H]cyclo(His-Pro) in adrenal, liver and kidney was demonstrated. Membrane preparations from adrenal and liver, but not from kidney, brain, pituitary, and other tissues were shown to bind cyclo(His-Pro) specifically.     

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in anuran (frog) skins

The distribution of cyclo (His-Pro)-like immunoreactivity in frog skins from seven frog species was examined. The chromatographic elution profile of cyclo (His-Pro)-like immunoreactivity in amphibian skins measured by radioimmunoassay corresponded precisely to that of [³H-Pro]-cyclo (His-Pro) after DEAE-Cellulose, Sephadex G-25 and high-pressure liquid chromatography. The concentrations of cyclo (His-Pro) in frog skins were much higher than the concentrations of TRH previously observed in skin and the concentrations of cyclo (His-Pro) in both brain and gastrointestinal tract.     

Morphone abstinence and quasi-abstinence effects after phosphodiesterase inhibitors and naloxone.

Naive or morphine-dependent rats received a single subcutaneous injection of a phosphodiesterase inhibitor; their behavioral responses were then recorded after a small subcutaneous dose of naloxone. In naive rats, the potent phosphidiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) produced acutely a state in which a small dose of naloxone (0.03 to 1.0 mg/kg subcutaneously) precipitated a quasi-morphine abstinence syndrome that was difficult to distinguish from the true abstinence syndrome, precipitated by the same dose of naloxone in rats made dependent on morphine. IBMX also intensified the true morphine abstinence syndrome. The potency with which IBMX, theophylline, caffeine and RO 20–1724 exerted these effects corresponded with their potency as inhibitors of cyclic-3′, 5′-AMP phosphodiesterase in rat brain homogenate. These and previous findings indicate that: (i) morphine-abstinence effects express increased activity of a central cyclic AMP mechanism; and (ii) naloxone can potently stimulate behavior in animals not treated with any opiate drug.     

Properties of histidyl-proline diketopiperazine [cyclo(His-Pro)] binding sites in rat liver

Characteristics of cyclo(His-Pro) binding sites in the rat liver were studied using 3H-labeled cyclo(His-Pro). Scatchard analysis suggested that the rat liver membrane had a single binding site with an apparent dissociation constant (Kd) of 7 X 10(-8) M. Pretreatment of membrane preparations with soybean trypsin inhibitor increased cyclo(His-Pro) binding, and the binding activity was sensitive to trypsin and phospholipase A digestion, suggesting that protein and phospholipid moieties are essential for cyclo(His-Pro) binding. Thiol reagents reduced binding activity, suggesting that the thiol group might be an important constituent of the cyclo(His-Pro) binding site. Cross-reactivities of TRH, TRH analogues, L-His and L-Pro were very low (0.2-9%). These findings indicate that specific binding sites for cyclo(His-Pro) in the rat liver have similar properties to the receptors for other polypeptides.     

A comparison of the inhibitory effects of clonidine and guanfacine on the behavioral and autonomic components of morphine withdrawal in rats

Intravenous administration of naloxone (0.5 mg/kg) to morphine dependent rats elicited classical autonomic and behavioral symptoms of narcotic abstinence including hypertension, tachycardia, withdrawal body shakes, escape attempts, diarrhea, etc. Pretreatment of dependent rats with either clonidine (3–90 μg/kg) or guanfacine (3–900 μg/kg) produced a dose-dependent reduction in the hypertensive response to subsequent injection of naloxone. Clonidine was about 12 times more potent than guanfacine in inhibiting this autonamic symptom of withdrawal. Both drugs were less effective at blocking body shakes and escapes, however, when all symptoms were combined in a ranked score, guanfacine was less effective than clonidine at reducing the ranked abstinence intensity score. Since clonidine blocked the autonomic component of withdrawal at doses more consistent with its clinical anti-withdrawal actions, it is possible that 1) measurement of behavioral signs of withdrawal in rats is a less sensitive index than is measurement of autonomic changes associated with withdrawal, or, 2) a reduction in autonomic outflow in general is most relevant to suppressing the apparent intensity of the abstinence syndrome.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

Baclofen reestablishes striatal and cortical dopamine concentrations during naloxone-precipitated withdrawal

The present study analyzes the effects of baclofen (BAC) on mice brain neurochemical alterations during the morphine (MOR) withdrawal syndrome. Male Swiss-Webster albino mice (27-33 g) were rendered dependent by intraperitoneal (i.p.) injection of MOR (2mg/kg), twice daily for 9 days. On day 10, the dependent animals were divided into two groups: one receiving naloxone (NAL; 6 mg/kg i.p.) to precipitate the withdrawal syndrome 60 min after the last dose of MOR and the other received BAC (2mg/kg, i.p.) followed by NAL (6 mg/kg, i.p.), injected 30 and 60 min after the last dose of MOR, respectively. Ten minutes after these treatments, mice were killed by decapitation and the striatum, cortex and hippocampus were dissected to determine endogenous concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites using HPLC with electrochemical detection. Striatal DA, dihydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) concentrations as well as cortical DA concentrations of the withdrawal groups decreased significantly with respect to the control groups. BAC attenuated the decrease in DA and DOPAC concentrations observed during the withdrawal, without modifying per se the control DA concentrations. No changes on 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) concentrations were observed during the MOR abstinence syndrome. The prevention caused by BAC on the decreased concentrations of DA induced by MOR withdrawal could have a therapeutic interest for the management of withdrawal syndrome.