Effects of thyrotropin-releasing hormone (TRH) and MK-771 on schedule-controlled behavior of squirrel monkeys, rabbits and pigeons

The effects of TRH (0.001-10.0 mg/kg) and a more potent TRH analog, MK-771 (0.001-5.6 mg/kg), were studied on comparable schedule-controlled performances of squirrel monkeys, rabbits and pigeons. Responding was maintained in the presence of different stimuli by a multiple fixed-ratio (FR), fixed-interval (FI) schedule of food presentation (monkeys and pigeons) or 0.25% saccharin solution (rabbits). Generally, TRH and MK-771 produced decreases in responding under both schedules and in all three species. TRH and MK-771 were roughly equipotent in the squirrel monkey, whereas in the pigeon and rabbit MK-771 was approximately 20 times more potent than TRH in decreasing responding to 50 percent of control levels. The duration of action of doses of TRH and MK-771 that reduced responding to 50 percent of control was approximately 3 hr in the squirrel monkey; recovery of performance occurred twice as fast under the FR schedules. With the pigeon, TRH effects that produced 50 percent decreases in responding lasted over 6 hours, whereas behaviorally comparable doses of MK-771 lasted about 4 hours. With few exceptions, TRH and MK-771 appear to produce similar effects of schedule-controlled behavioral performances of the squirrel monkey, rabbit and pigeon. Compared to the effects of other behaviorally-active substances under these procedures, TRH and MK-771 exert a distinctive array of effects.     

Increase of tonic parasympathetic outflow to the pupil produced by an analog of TRH (MK-771)

MK-771, a potent analog of thyrotropin-releasing, hormone, was found to cause a long-lasting, dose-dependent antagonism of the centrally induced pupillary dilation produced by clonidine in anesthetized cats. MK-771 had little direct effect on the dilated iris in cats treated with mecamylamine. Both MK-771 and TRH selectively antagonized the pupillary dilation, but not the contractions of the nicitating membrane induced by peripheral sympathetic nerve stimulation. MK-771 was found to increase nerve activity in the short ciliary nerve. TRH, although studied less extensively, produced similar effects. This action of MK-771 was not blocked by prior sectioning of the spinal cord, by elimination of the tonic light reflex pathway, or by treatment with scopolamine or atropine. These findings suggest that the pupillary effects of MK-771 (and TRH) are related to the ability to increase the parasympathetic tone to the iris.     

TRH analog MK-771 reverses neurochemical and learning deficits in medial septal-lesioned rats

Microinjection of ibotenic acid into medial septum of rats decreased choline acetyltransferase (CAT) and high-affinity choline uptake (HACU) activities in hippocampus and retarded the learning of a spatial memory task in the radial-arm maze. Administration of MK-771, a stable TRH analog, to such animals restored HACU activity in hippocampus to normal levels. Daily treatment of rats with MK-771 prior to maze running also restored the animals' learning ability. MK-771 did not enhance hippocampal HACU activity or maze performance in sham-lesioned rats. These results suggest that MK-771 reversed the ibotenic acid-induced memory deficit by restoring septohippocampal cholinergic function. MK-771 and other TRH analogs may represent novel agents for improving memory deficits produced by cholinergic insufficiency in Alzheimer's disease.     

Substance P antagonist-induced spinal cord vasoconstriction: Effects of thyrotropin-releasing hormone and substance P agonists

Local spinal cord vasomotor effects of 3 substance P (SP) antagonists were studied in the rat following intrathecal (IT) administration. Each SP antagonist (3.3 nmol) increased spinal cord vascular resistance and reduced blood flow. A LH-RH antagonist analog (10 nmol) of similar molecular weight and which also contained multiple D-Trp residues did not cause spinal cord vasoconstriction. The vasoconstrictor action of the SP antagonist, [D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹]-SP ([D-Arg]-SP) was unaffected by pretreatment with a stable SP receptor agonist (5 nmol IT). Given evidence for a cerebral vasodilator action of TRH agonists, the effects of TRH (IV) and a stable TRH analog (MK-771, IT) on [D-Arg]-SP-induced vasoconstriction were also assessed. Neither TRH nor MK-771 prevented the [D-Arg]-SP-induced vasoconstriction. However, TRH (IV) but not MK-771 (IT) partially opposed [D-Arg]-SP-induced reduction in thoracic spinal cord blood flow. Thus, SP antagonists cause spinal cord vasoconstriction by a non-SP receptor mediated phenomenon. In addition, the attenuation of SP-antagonist-induced neuropathological changes previously reported with IV. TRH administration is likely due to less severe consequences of vasoconstriction in the presence of a higher initial baseline blood flow rather than direct prevention of the vasoconstriction.     

Thyrotropin-Releasing Hormone and Its Analog MK-771 Increase the Cerebroventricular Perfusate Content of Dihydroxyphenylacetic Acid

The effects of thyrotropin-releasing hormone (TRH) and its synthetic analog, pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide (MK-771), were determined on the efflux of dihydroxyphenylacetic acid (DOPAC) collected from push-pull cannulae chronically implanted into the lateral cerebral ventricles of rats. Intracerebroventricular and intraperitoneal injections of both peptides increased the efflux of DOPAC. These results suggest that TRH and MK-771 increase the activity of dopaminergic neurons that terminate in periventricular regions.     

Propranolol and methylatropine antagonize the cardiovascular effects produced by microinjection of the TRH analog MK-771 into the preoptic suprachiasmatic nucleus

Thyrotropin-releasing hormone (TRH) has been shown to increase heart rate as well as blood pressure when administered into rat brain. The present study investigated the mechanism by which the TRH analog MK-771 produces these effects when injected into the preoptic suprachiasmatic nucleus (POSC). MK-771, at a dose of 125 pmol (50 ng), produced significant increases in both heart rate and blood pressure. These effects occurred within 5 minutes of microinjection and lasted approximately 20-30 minutes. Pretreatment with either the beta-adrenergic antagonist propranolol or the muscarinic antagonist methylatropine, administered into the POSC, significantly altered the response produced by MK-771. Propranolol, at a dose of 7 nmol, and methylatropine at a dose of 0.5 nmol, significantly inhibited the tachycardia produced by MK-771. In addition, methylatropine, at a dose of 0.5 nmol, significantly reduced the increase in diastolic pressure produced by the TRH agonist. These results are consistent with the idea that TRH agonists, when administered centrally, produce cardiovascular alterations through the autonomic nervous system.     

Analogs of thyrotropin-releasing hormone (TRH): Receptor affinities in brains,spinal cords,and pituitaries of different species

[³H](3-Me-His²) thyrotropin-releasing hormone ([³H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, K_ds=5–9 nM; n=3–4) but to different densities of these sites (B _max range 6–145 fmol/mg protein) (rabbit SC>sheep PITG.pig brain>dog brain>rat brain>bovine and dog PIT). Various TRH analogs competitively inhibited [³H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH>TRH> CG3703RX77368MK-771>TRH Glycinamide>Glu¹-TRHCG3509NVal²-TRH>>>TRH free acid>>>and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [³H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and 3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.     

Effects of thyrotropin releasing hormone and its analogues on unconsciousness following head injury in mice.

We examined the effects of thyrotropin releasing hormone (TRH) and its analogues (DN-1417: gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; MK-771: L-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide; TSII-37: H-Lys-Gln-His-Pro-Gly-Ser-OH) on arousal in head injured mice, an animal model of unconsciousness. TRH, DN-1417, MK-771 and TSII-37 were injected 10 min before the head injury. TRH, DN-1417 and MK-771 caused dose-dependent decreases in the time required for recovery of the righting reflex time and in the time from the head injury to the onset of spontaneous movement. TSII-37 had no effect, when compared with the control group. In terms of the minimum effective dose, TRH and DN-1417 were of similar potency, but MK-771 was about 30-fold stronger than TRH. Measurement of the cross-reactivities of these TRH analogues by radiolabeled receptor assay suggest that the structure-binding relationship is proportional to the structure-activity relationship.     

Potential anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues

The anti-depressive effects of thyrotropin releasing hormone (TRH) and its analogues (DN-1417: gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate; MK-771: L-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) were examined in behavioral despair rats, an animal model of depression. TRH, DN-1417, MK-771, amitriptyline and diazepam were injected three times after the first forced swimming. One hr after the last injection, a 5-min swimming test was performed. Experimental animals were placed in a Hall's type open-field apparatus immediately before and after the 5-min test, and their locomotor activities were determined. No significant difference was noted in the locomotor activity immediately before the 5-min test among any group. In the 5-min swimming test, TRH, DN-1417 and MK-771 caused a dose-dependent decrease in immobility, showing an anti-depressive effect similar to amitriptyline. Diazepam showed no difference compared with the control group. After the swimming test, locomotor activity remarkably decreased in the control rats, while decreased locomotor activity was partially prevented in the TRH, DN-1417, MK-771 and amitriptyline treated rats which exhibited active movement not only during the swimming period but also after it. In terms of the minimum effective dose, TRH and DN-1417 seemed to be of similar potency, while MK-771 was 40-fold stronger than TRH. An examination of a possible correlation between the cross-reactivity of TRH analogues in a radioreceptor assay and the effects of the analogues on despair rats suggested that the structure-binding relationship was proportional to the structure-activity relationship.     

Thyrotropin releasing hormone prevents abnormalities of cortical acetylcholine and monoamines in mice following head injury

We investigated the effects of thyrotropin releasing hormone (TRH) on changes in cortical concentrations of acetylcholine (ACh) and monoamines produced by concussion in mice. Concussion was induced by dropping a metal rod on the head, and the concentration of ACh, norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the cerebral cortex were measured by HPLC. We also examined the arousal effects of 0.5 mg/kg of TRH and 0.015 mg/kg of -pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide (MK-771), a TRH analogue, injected intraperitoneally 10 min before concussion, on neurotransmitter concentrations. Mice were sacrificed at 25 (representing the righting reflex time) and 210 s (representing spontaneous movement time). At 25 s after concussion, the concentration of ACh was significantly higher than in control mice, but pretreatment with TRH and MK-771 prevented the rise in ACh. In contrast, head injury significantly reduced NE concentration. TRH and MK-771 also prevented the fall in NE. Concussion did not change cortical concentrations of DA and 5-HT. Our results suggest that disturbances of consciousness produced by concussion may be due to increased ACh and diminished NE in the cerebral cortex. Our findings also suggest that the arousal effects of TRH on concussion-induced disturbances of consciousness are due to normalization of cortical cholinergic and noradrenergic neuronal systems.     

Effects of thyrotropin-releasing hormone (TRH) and MK-771 on behavior of squirrel monkeys controlled by noxious stimuli

The effects of TRH (0.1-30 mg/kg) and an enzyme-resistant analogue, MK-771 (0.1-10 mg/kg), were characterized in squirrel monkeys on responding maintained in the presence of different visual stimuli by a multiple 3-min fixed-interval (FI), 30-response fixed-ratio (FR) schedule of stimulus-shock termination or by a multiple 5-min FI schedule of food or shock presentation. Under the termination schedule, the first response at the end of 3 min in the FI component or the completion of the 30-response requirement in the FR component terminated the visual stimulus in the presence of which shocks occurred (escape schedule). Under the schedule of food or shock presentation, the first response at the end of the 5-min FI produced food in the presence of red stimulus lights or shock in the presence of white lights. TRH and MK-771 produced large, dose-related increases in responding maintained under the FR stimulus-shock termination schedule whereas these peptides produced smaller increases or did not affect responding under the FI schedule. TRH and MK-771 also produced marked increases in responding maintained by shock presentation at doses that did not alter or decreased food-maintained responding in the same subject. Thus, performances maintained by noxious stimuli are uniquely sensitive to the rate-increasing effects of TRH and MK-771. These findings suggest that the behavioral effects of the neuropeptides, TRH and MK-771, can depend on the specific consequences of behavior and, as such, the effects of these substances are determined by many of the same variables that determine the effects of other behaviorally-active drugs.     

The effect of TRH and a related tripeptide, L-N-(2-oxopiperidin-6-yl-carbonyl)-L-histidyl-L-thiazolidine-4-carboxamide (MK-771, OHT), on the depressant action of barbiturates and alcohol in mice and rats.

TRH and a related tripeptide, L-N(2-oxopiperidin-6-ylcarbonyl)-L-histidyl-L-thiazolidine-4-carboxamide (MK-771, OHT), shortened pentobarbital sleeping time of mice in a dose-related fashion. The regression lines were not parallel, so that a strict potency comparison cannot be made between the two compounds. However, comparison between the doses of TRH and OHT required to achieve a maximum response showed the latter to be some 100 times as potent as the former. TRH and OHT shortened methohexital sleeping time of rats and interfered with pentobarbital-induced and alcohol-induced hypothermia in mice. The depressant effect of alcohol upon electroshock escape, rotorod performance, and achievement of a simple, learned task was partly overcome by TRH and OHT. Although quantitative comparisons were not made in all tests, OHT was consistently the more potent of the two compounds.     

Neurotensin-induced antinociception and hypothermia in mice: antagonism by TRH and structural analogs of TRH

Intracisternal (IC) administration of neurotensin (NT) in a dose of 10 micrograms produced a significant hypothermia and antinociception in the hot-plate test in mice. Both of these effects of IC NT were completely antagonized by concomitant administration of equimolar doses of thyrotropin-releasing hormone (TRH) and several TRH congeners including 3-methyl-His-TRH (pGlu-3-methyl-His-Pro-NH2), MK-771 (pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide), beta-ala-TRH (pGlu-His-Pro-beta-ala-NH2), and RX-77368 (pGlu-His-dimethyl-Pro-NH2). The antagonism by TRH and TRH analogs on NT-induced hypothermia and antinociception was dose-dependent. Of particular interest was the finding that RX-77368 not only blocked the effects of NT but also produced hyperalgesia. It appears that TRH analogs that are more resistant to biologic degradation are, like TRH, capable of blocking NT-induced behaviors.     

Two novel thioamide analogues of TRH with selective activity on CNS.

TRH analogues containing C-terminal tioamide group and norvaline ([Nva2, Prot3] TRH) or norleucine ([Nle2, Prot3] TRH) in position 2 were synthesized and tested for hormonal and central nervous system (CNS) activities. Receptor binding studies revealed that the analogues neither bind to pituitary nor to brain TRH receptors. Accordingly, no TSH releasing activity was recorded. However, both analogues significantly affected sleeping time and breathing frequency. Dissociation of endocrine effects from those on the CNS of [Prot3] TRH was achieved with the replacement of histidine2 by aliphatic amino acids. The presence of central histidine is not essential for the analogues to be active on the CNS.     

Lack of Usefulness of DN-1417 for Characterization of a CNS Receptor for Thyrotropin-Releasing Hormone

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of MK-771, A TRH analog,on pentobarbital-induced alterations of cholinergic parameters in discrete regions of rat brain

MK-771 (l-pyro-2-aminoadipyl-histidyl-thiazolidine-4-carboxamide) was administered intraventricularly to conscious and pentobarbital-narcotized rats. In the conscious rats MK-771 did not affect the regional levels of acetylcholine (ACh) or the rate of sodium-dependent high-affinity choline uptake (HACU). MK-771 was found to antagonize pentobarbital-induced elevations of ACh levels in the cortex, hippocampus and striatum. MK-771 also reversed the depressant effects of pentobarbital on the HACU of the cortex and hippocampus. Striatal HACU was unaltered by the administration of pentobarbital or the combination of pentobarbital and MK-771.     

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.     

Thyrotropin releasing hormone: autonomic effects upon cardiorespiratory function in endotoxic shock

These studies define potential sites and mechanisms by which thyrotropin releasing hormone (TRH) stimulates cardiorespiratory function in normotensive rats as well as in rats subjected to endotoxic shock. Changes in mean arterial pressure, pulse pressure, heart rate, and respiratory rate were determined in conscious animals following injection of TRH into the lateral, third, or fourth ventricular spaces. Injections of TRH into the third ventricular space resulted in a greater increase in cardiorespiratory variables than did fourth ventricular injection. In endotoxin-treated rats, the cardiorespiratory effects of intracerebroventricular (icv) TRH and its analog MK 771 were assessed. TRH and MK 771 were shown to act within the brain to reverse endotoxic shock hypotension; at the doses used, the pressor effects of these two tripeptides were achieved through selectively different actions upon heart rate and pulse pressure. Adrenal demedullated and sham-operated control rats subjected to endotoxic shock were injected with icv and intravenous (iv) TRH in order to evaluate the potential involvement of sympatho-medullary function in cardiorespiratory responses. The cardiovascular effects of icv TRH were dependent upon adrenal medullary integrity; effects of iv TRH were not. Doses of iv TRH which effectively reverse shock neither altered nociceptive latencies nor interfered with analgesic responses to morphine. Collectively, these studies reinforce the potential therapeutic utility of TRH and its analogs in the treatment of shock and indicate potential sites and mechanisms which mediate these salutary effects.     

Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.     

Biological activities of thionated thyrotropin-releasing hormone analogs.

Analogs of thyrotropin-releasing hormone (Glp-His-Pro-NH2, TRH) have been prepared which contain thioamide moieties in the pyroglutamic acid ring, the carboxyamide proline terminus, and in both positions (dithio). These compounds have been tested for TSH-releasing activities (in vitro and in vivo), and for binding to TRH receptors in rat pituitary and cortex. The monothionated analogs showed no significant differences in TSH-releasing potency from TRH either in vitro or in vivo. However, with two thioamide replacements the potency decreases about 50%. Significantly, in terms of receptor selectivity, thionation has resulted in differentiation between brain receptors (pituitary and cortex). The Pro psi[CSNH2] and dithio analogs were more selective (higher affinity to pituitary receptors) than the parent hormone, while the analog containing a thioamide replacement in the pyroglutamyl ring had lower affinity and was not selective. These results suggest that the subtle exchange of sulphur for oxygen can have an important impact on both receptor selectivity and affinity within a biologically active peptide.