Differences in the pharmacological actions of intrathecally administered neurotensin and morphine

Neurotensin or morphine can each cause hypothermia and an antinocisponsive effect when administered into the liquor spaces of the rat brain. These actions of neurotensin are not blocked by naloxone whereas those of morphine are. The present experiments were carried out to examine the action of each substance following its injection into the subarachnoid space of the spinal cord. Given intrathecally, neurotensin evoked a dose-related fall in the rectal temperature of the rat without exerting an antinocisponsive action. Morphine on the other hand evoked hyperthermia and a dose-related antinocisponsive action. Since neurotensin exerted an effect on rectal temperature opposite to that of morphine and failed to exert an antinocisponsive effect, the data provide further evidence to suggest that neurotensin and morphine exert their effect via different mechanisms. Furthermore, the results also suggest that neurotensin exerts its antinocisponsive action via a supraspinal site.     

Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.     

Regulation of excitation-secretion coupling by thyrotropin-releasing hormone (TRH): Evidence for TRH receptor-ion channel coupling in cultured pituitary cells

Summary The electrophysiological and secretory properties of a well-studied clonal line of rat anterior pituitary cells (GH₃) have been compared with a new line of morphologically distinct cells derived from it (XG-10). The properties of the latter cells differ from the parent cells in that they do not have receptors for thyrotropin-releasing hormone and their basal rate of secretion is substantially higher (ca. three- to fivefold). While both cell types generate Ca⁺⁺ spikes, the duration of the spike in XG-10 cells (ca. 500 msec) is about 2 orders of magnitude longer than that in GH₃ cells (5–10 msec). The current-voltage characteristics of the two cell types are markedly different; the conductance of GH₃ cells is at least 20-fold higher than XG-10 cells when cells are depolarized to more positive potentials than the threshold for Ca⁺⁺ spikes (–35 mV). While treatment of GH₃ cells with the secretagogues tetraethylammonium chloride or thyrotropin-releasing hormone decreases the conductance in this voltage region to approximately the same as that for XG-10 cells, the electrophysiological and secretory properties of XG-10 cells are unaffected by treatment with either of these agents. Results of this comparative study suggest that XG-10 cells lack tetraethylammonium-sensitive K⁺ channels. The parallel loss of thyrotropin-releasing hormone receptor binding activity and of a K⁺ channel in XG-10 cells implies that the thyrotropin-releasing hormone receptor may be coupled with, or be an integral part of, this channel. Apparently thyrotropin-releasing hormone, like tetraethylammonium chloride, acts by inhibiting K⁺ channels resulting in a prolongation of the action potential, promoting Ca⁺⁺ influx and subsequently enhancing hormone secretion.     

Effects of mammalian and avian neurotensins and neurotensin fragments on wet-dog shaking and body temperature in the rat

The ability of mammalian and avian neurotensins and some neurotensin fragments to reduce wet-dog shaking (WDS) induced by thyrotrophin-releasing hormone (TRH) and to influence rectal temperature was tested after their injection into the periaqueductal grey region of male rats. Both neurotensins inhibited TRH-induced WDS and reduced rectal temperature by 2 degrees C; this latter effect was prevented by prior TRH administration. Of the four neurotensin fragments tested, both (1-8)- and (8-13)-neurotensin reduced WDS but only (8-13)-neurotensin reduced rectal temperature significantly. (1-6)- and (1-11)-neurotensin were without effect in either test system. From the activity of the various peptides, further examples of the mutual antagonism between TRH and neurotensin have been demonstrated. It is suggested that there is a possible role for neurotensin in controlling body temperature via the periaqueductal grey and that this may be one function of neurotensin in avian species; there may also be more than one receptor system binding neurotensin in the brain.     

Involvement of thyrotropin-releasing hormone (TRH) neural system of the brain in pentylenetetrazol-induced seizures

In order to study the relationship between pentylenetetrazol (PTZ)-induced seizures and the thyrotropin-releasing hormone (TRH) neural system, immunoreactive TRH (IR-TRH) and TRH receptor binding activity were determined in discrete regions of the rat brain before as well as 40 s (immediately before seizures), 150 s (during seizures) and 24 h after an intraperitoneal injection of PTZ (75 mg/kg). IR-TRH markedly increased in the septum 40 and 150 s after the injection, and also in the hippocampus and the thalamus-midbrain region 40 and 150 s after the injection, respectively. However, no significant changes were observed in the TRH receptor binding before, during or after the seizures, suggesting that the increased IR-TRH was not released into the synaptic cleft. This speculation was supported by the dose-dependent inhibition of PTZ-induced generalized seizures by the pre-treatment with TRH or its analogue DN-1417 into the cerebral ventricle.     

The TRH neuronal phenotype forms embryonic cell clusters that go on to establish a regionalized cell fate in forebrain

How neurons diversify in developing brain to produce discrete cell fates in their appropriate regions remains a fundamental question. Embryonic Xenopus was previously used to identify juxtaposed embryonic cells that first express proopiomelanocortin mRNA in forebrain and pituitary, supporting the idea that this neuropeptide phenotype is induced locally. (Hayes and Loh, 1990, Development 110:747–757). To begin to examine how a more widespread population of forebrain cells is set up, the present focus is on the thyrotropin-releasing hormone (TRH) phenotype. Serial section in situ hybridization histochemistry produced the unexpected finding that the adult-like TRH system spanning forebrain and comprising over six different telencephalic and diencephalic nuclei, is preceded by an embryonic TRH cell population that is initially localized and then highly regionalized in the area from which the adult pattern develops. Thus, the first TRH cells, detected in vivo after 35 h (stage 29/30), were confined to discrete anterior or posterior bilateral clusters in embryonic forebrain or hindbrain. Thereafter, the TRH cell clusters in diencephelon, but not hindbrain, expanded to form rows, extending anteriorly into telencephalon and bifurcating posteriorly around the infundibulum. By 80 h (stage 42), after extensive brain morphogenesis, these forebrain rows showed regional differences in levels of TRH and mRNA corresponding to the specific brain nuclei that have been shown to contain TRH cells in adult. These findings show that subsets of phenotype-specific forebrain cell first form a regionalized neuronal cell fate before distinct brain nuclei form. This is turn points to the testable hypothesis in Xenopus that certain neuronal cell fates in forebrain may be dictated by cell lineage or local induction. 1994 John Wiley & Sons, Inc.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Daily torpor in mice: high foraging costs trigger energy-saving hypothermia

Many animal species employ natural hypothermia in seasonal (hibernation) and daily (torpor) strategies to save energy. Facultative daily torpor is a typical response to fluctuations in food availability, but the relationship between environmental quality, foraging behaviour and torpor responses is poorly understood. We studied body temperature responses of outbred ICR (CD-1) mice exposed to different food reward schedules, simulating variation in habitat quality. Our main comparison was between female mice exposed to low foraging-cost environments and high-cost environments. As controls, we pair-fed a group of inactive animals (no-cost treatment) the same amount of pellets as high-cost animals. Mice faced with high foraging costs were more likely to employ torpor than mice exposed to low foraging costs, or no-cost controls (100% versus 40% and 33% of animals, respectively). While resting-phase temperature showed a non-significant decrease in high-cost animals, torpor was not associated with depressions in active-phase body temperature. These results demonstrate (i) that mice show daily torpor in response to poor foraging conditions; (ii) that torpor incidence is not attributable to food restriction alone; and (iii) that high levels of nocturnal activity do not preclude the use of daily torpor as an energy-saving strategy. The finding that daily torpor is not restricted to conditions of severe starvation puts torpor in mice in a more fundamental ecological context.     

Interaction of neurotensin with prostaglandin E2 and prostaglandin synthesis inhibitors: effects on colonic temperature in mice

Neurotensin (NT) administered intracisternally (i.c.) to adult mice produced a marked hypothermia while prostaglandin E₂, administered by the same route, produced hyperthermia. When administered concurrently the effects of the two substances were neutralized. The prostaglandin synthesis inhibitors, indomethacin and acetylsalicylic acid, were injected subcutaneously 30 min prior to i.c. administered NT and/or thyrotropin-releasing hormone (TRH). Both inhibitors failed to potentiate the hypothermia induced by NT or alter its antagonism by TRH in mice kept at 26°C. When mice were kept at 6°C, pretreatment with indomethacin, but not acetylsalicylic acid, potentiated NT-induced hypothermia and prevented its antagonism by TRH. Because indomethacin inhibits synthesis of prostaglandins within the central nervous system (CNS) as well as in peripheral organs while acetylsalicylic acid acts only in the periphery, it appears that NT-induced hypothermia in a cold environment is enhanced by a reduction of prostaglandins in the CNS.     

Binding of copper(II) to thyrotropin-releasing hormone (TRH) and its analogs

Spectroscopy (UV-Vis, ¹H NMR, ESR) and electrochemistry revealed details of the structure of the Cu(II)-TRH (pyroglutamyl-histidyl-prolyl amide) complex. The ¹H NMR spectrum of TRH has been assigned. NMR spectra of TRH in the presence of Cu(II) showed that Cu(II) initially binds TRH through the imidazole. TRH analogs, pGlu-His-Pro-OH, pGlu-(1-Me)His-Pro-amide, pGlu-His-(3,4-dehydro)Pro-amide, pGlu-His-OH, pGlu-Glu-Pro-amide, and pGlu-Phe-Pro-amide provided comparison data. The stoichiometry of the major Cu(II)-TRH complex at pH 7.45 and greater is 1:1. The conditional formation constant (in pH 9.84 borate with 12.0 mM tartrate) for the formation of the complex is above 10⁵ M⁻¹. The coordination starts from the 1-N of the histidyl imidazole, and then proceeds along the backbone involving the deprotonated pGlu-His amide and the lactam nitrogen of the pGlu residue. The fourth equatorial donor is an oxygen donor from water. Hydroxide begins to replace the water before the pH reaches 11. Minority species with stoichiometry of Cu-(TRH)_x (x = 2-4) probably exist at pH lower than 8.0. In non-buffered aqueous solutions, TRH acts as a monodentate ligand and forms a Cu(II)-(TRH)₄ complex through imidazole nitrogens. All the His-containing analogs behave like TRH in terms of the above properties.     

Supraspinal circuitry mediating opioid antinociception: Antagonist and synergy studies in multiple sites

Supraspinal opioid antinociception is mediated by sensitive brain sites capable of supporting this response following microinjection of opioid agonists. These sites include the ventrolateral periaqueductal gray (vIPAG), the rostral ventromedial medulla (RVM), the locus coeruleus and the amygdala. Each of these sites comprise an interconnected anatomical and physiologically relevant system mediating antinociceptive responses through regional interactions. Such interactions have been identified using two pharmacological approaches: (1) the ability of selective antagonists delivered to one site to block antinociception elicited by opioid agonists in a second site, and (2) the presence of synergistic antinociceptive interactions following simultaneous administration of subthreshold doses of opioid agonists into pairs of sites. Thus, the RVM has essential serotonergic, opioid, cholinergic and NMDA synapses that are necessary for the full expression of morphine antinociception elicited from the vIPAG, and the vIPAG has essential opioid synapses that are necessary for the full expression of opioid antinociception elicited from the amygdala. Further, the vIPAG, RVM, locus coeruleus and amygdala interact with each other in synergistically supporting opioid antinociception.     

Dopamine D2-receptors mediate hypothermia in mice: ICV and IP effects of agonists and antagonists

The effect of centrally and peripherally administered dopamine D1 and D2 specific compounds on core body temperature in mice was investigated. Quinpirole (LY-17155), a D2 agonist, induced a dose-dependent fall in body temperature (2.4–11.6%; p<0.003) when injected intraperitoneally (ip, 0.3–3.0 mg/kg) and intracerebroventricularly (icv, 0.1 mg/kg). This quinpirole-induced (1.0 mg/kg, ip) hypothermia was reversed by the central and peripheral administration of the D2 antagonists S-(–)-sulpiride (3.0–30.0 mg/kg, ip; 0.1–3.0 mg/kg, icv) and spiperone (0.03 and 0.1 mg/kg, ip; 0.03–3.0 mg/kg, icv). Domperidone, a D2 antagonist which does not cross the blood brain barrier, had no effect on quinpirole-induced hypothermia (1.0–10.0 mg/kg, ip). Domperidone partially reversed quinpirole-induced hypothermia at 0.1–30.0 mg/kg, icv. The D1 agonist, SKF-38393 at a high dose of 10.0 mg/kg, ip mildly attenuated quinpirole-induced hypothermia (a 1.8% increase in temperature). SKF-38393 at 10.0 mg/kg, icv potentiated quinpirole-induced hypothermia. SCH-23390 (0.1–3.0 mg/kg, ip), a D1 antagonist, had no effect on quinpirole-induced hypothermia and potentiated the hypothermia when administered icv. An ineffective icv dose of spiperone (0.01 mg/kg) in reversing quinpirole-induced hypothermia was rendered effective by prior administration of SCH-23390 (0.1–3.0 mg/kg, icv) but not by SKF-38393 (1.0–10.0 mg/kg, icv). These data suggest a central D2 receptor mechanism mediating hypothermia in mice which is capable of being modulated by the D1 receptor.     

Mu-opiate binding and morphine antagonism by octapeptide analogs of somatostatin

A series of cyclic conformationally restrained octapeptide analogs of somatostatin were examined for their ability to inhibit the binding of tritiated mu, kappa, and delta opiate receptor ligands. Several of these substances were found to have high affinity for mu opiate receptors while having very low affinity for both kappa and delta receptors. Previous suggestions that somatostatin analogs exhibit opiate antagonist activity led to a study of the ability of the two most potent compounds to inhibit morphine analgesia in rats after intracerebroventricular injection. One of the compounds significantly antagonized morphine analgesia although the other displayed severe toxicity. These two compounds differed in that the very toxic compound had previously been found to possess significant somatostatin activity. It thus appears that the structural requirements for toxicity and somatostatin activity can be differentiated from those for opiate activity.     

Ethanol-induced hypothermia and ethanol consumption in the rat are affected by low-level microwave irradiation

Microwave irradiation of rats by circularly polarized, 2,450-MHz, pulsed waves (2-μs pulses; 500 pps) was performed in waveguides to determine effects on ethanol-induced hypothermia and on ethanol consumption. Rats injected intraperitoneally with ethanol (3 g/kg in a 25% v/v water solution) immediately after 45 min of microwave irradiation exhibited attenuation of the initial rate of fall in body temperature, which was elicited by the ethanol, but exhibited no significant difference in maximal hypothermia as compared with that of sham-irradiated rats. Microwave irradiation did not affect the consumption of a 10% sucrose (w/v) solution by water-deprived rats. However, it enhanced the consumption of a solution of 10% sucrose (w/v) + 15% ethanol (v/v) by water-deprived animals. These results were obtained at a specific absorption rate (SAR) of 0.6 W/kg, which rate of energy dosing would require a power density of 3–6 mW/cm² if exposure of the animals had occurred to a 12-cm plane wave.     

Central administration of thyrotropin-releasing hormone and histidyl-proline-diketopiperazine disrupts the acquisition of a food rewarded task by a non-aversive action

The effects of thyrotropin-releasing hormone (TRH) and its metabolites on operant behaviour have rarely been explored. In this study, the effects of intracerebroventricular (icv) administration of TRH and histidyl-proline-diketopiperazine (DKP), a metabolite of TRH, on the acquisition of a food-rewarded lever-press task were compared with saline-treated controls. TRH and DKP severely retarded the acquisition of lever pressing. The effects of systemically administered D-amphetamine were also examined in order to test whether this result was due to any stimulant properties of these peptides. These results suggest that stimulatory effects do not adequately account for impaired acquisition. The possibility that the disruption of learning was due to an aversive effect of icv administration of these peptides was tested by means of a conditioned place paradigm. Neither peptide induced an avoidance of the environment with which it had previously been paired. Several possible reasons for the peptides' adverse effect on learning are discussed, including the possibility that TRH and DKP act on attentional mechanisms.     

Asymmetry of modifications of the electrical activity of the rat cortex induced by intraventricular injections of thyrotropin-releasing hormone

We studied modifications in the mass electrical activity of the cortex (ECoG) induced by injections of thyrotropin-releasing hormone (TRH) into the left or right lateral brain ventricle in rats kept under conditions close to free behavior. It was found that these effects are characterized by a significant interhemisphere asymmetry. We postulate that the pharmacological (in particular, antidepressive) effects of TRH are related to its ability to intensify inhibitory processes in the left cerebral hemisphere and activating processes in the right hemisphere.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 386–390, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

不同性别及性腺功能对梭曼引起大鼠低温的影响

目的：研究不同性别及性腺功能对梭曼引起大鼠低温的影响。方法：用数字体温计测量大鼠的结肠温度,观察梭曼引起正常雄性和雌性大鼠低温反应的性别差异以及切除性腺后对其作用的影响。结果：①雌性大鼠对梭曼引起的低温反应比雄鼠更敏感。②切除雄性大鼠睾丸后能明显提高对梭曼低温反应的敏感性,而切除卵巢的雌性大鼠对梭曼的低温反应与模拟手术组无明显差异。结论：雄性和雌性大鼠对梭曼敏感性的性别差异主要取决于睾丸的功能。     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

FasL shedding is reduced by hypothermia in experimental stroke

Protection by mild hypothermia has previously been associated with better mitochondrial preservation and suppression of the intrinsic apoptotic pathway. It is also known that the brain may undergo apoptotic death via extrinsic, or receptor-mediated pathways, such as that triggered by Fas/FasL. Male Sprague-Dawley rats subjected to 2 h middle cerebral artery occlusion with 2 h intraischemic mild hypothermia (33°C) were assayed for Fas, FasL and caspase-8 expression. Ischemia increased Fas, but decreased FasL by ∼ 50–60% at 6 and 24 h post-insult. Mild hypothermia significantly reduced expression of Fas and processed caspase-8 both by ∼ 50%, but prevented ischemia-induced FasL decreases. Fractionation revealed that soluble/shed FasL (sFasL) was decreased by hypothermia, while membrane-bound FasL (mFasL) increased. To more directly assess the significance of the Fas/FasL pathway in ischemic stroke, primary neuron cultures were exposed to oxygen glucose deprivation. Since FasL is cleaved by matrix metalloproteinases (MMPs), and mild hypothermia decreases MMP expression, treatment with a pan-MMP inhibitor also decreased sFasL. Thus, mild hypothermia is associated with reduced Fas expression and caspase-8 activation. Hypothermia prevented total FasL decreases, and most of it remained membrane-bound. These findings reveal new observations regarding the effect of mild hypothermia on the Fas/FasL and MMP systems.