Virender M. Labroo

Differential effect of fluorinated analogs of TRH on the cardiovascular system and prolactin release

The effects of thyrotropin-releasing hormone (TRH) and the TRH-analogs, 4-fluoro-Im-TRH (4-F-TRH) and 2-trifluoromethyl-Im-TRH (2-TFM-TRH), on the cardiovascular system and prolactin (PRL) release were examined in conscious rats. TRH (2.8 or 28 nmol) injected into the anterior hypothalamus produced dose-dependent increments in blood pressure and heart rate; plasma PRL was increased twofold after the higher dose of TRH. 4-F-TRH had effects similar to those of TRH on both the cardiovascular and PRL response. In contrast, the 2-TFM-TRH was significantly less active than TRH or 4-F-TRH in eliciting tachycardia, yet was noticeably more potent in affecting PRL release. These data suggest that the receptors for TRH-induced PRL release may be different from TRH-receptors which mediate central cardiovascular responses.

First direct fluorination of tyrosine-containing biologically active peptides

Abstract Using acetyl hypoflurite a regiospecific electrophilic fluorination of the tyrosine ring of the N-terminal tetrapeptide amide (Tyr-D-Ala-Phe-Gly-NH 2 ) sequence of the opiate peptide dermorphin has been achieved in good yields.

Receptor binding of fluorinated histidine analogs of thyrotropin-releasing hormone in various regions of the rat brain

Binding properties of [4(5)-fluoro-imidazole-His2]-TRH (4(5)-F-TRH), [2-trifluoromethyl-imidazole-His2]-TRH (2-CF3-TRH) and [4(5)-trifluoromethyl-imidazole-His2]-TRH (4(5)-CF3-TRH), three novel TRH analogs, have been evaluated in rat pituitary, hypothalamus, brainstem and cortex tissue. 4(5)-F-TRH, previously shown to elicit arterial pressor responses and prolactin release similar to those of TRH, binds to TRH receptors with low, micromolar affinity (Ki = 7.5-13.5 microM). 2-CF3-TRH, an analog of less cardiovascular but increased prolactin-releasing activity, shows Ki values of 3.3-4.9 microM. 4(5)-CF3-TRH, which shows comparable biological activity to 2-CF3-TRH, demonstrates a binding affinity which is virtually nonspecific (Ki = 0.39-1.01 mM). It is therefore concluded that the biological effects of these analogs are mediated either through low affinity TRH binding sites not recognized by [3H][3Me-His2]-TRH or through mechanisms not involving TRH receptors as such.

Norvaline2-TRH : binding to TRH receptors in rat brain homogenates

Norvaline2-thyrotropin-releasing hormone ([Nva2]TRH) has been described as a thyrotropin-releasing hormone (TRH) analog with no thyrotropin (TSH)-releasing capacity but enhanced analeptic activity compared with TRH, as shown by the reversal of haloperidol-induced catalepsy. We have evaluated the receptor-binding properties of [Nva2]TRH in homogenates of rat anterior pituitary, hypothalamus, brainstem and cortex tissue, using [3H]TRH and [3H][3-Me-His2]TRH as radioligands. Apparent Ki values at high affinity TRH-binding sites, labelled predominantly by [3H][3-Me-His2]TRH, ranged from 17.0 to 36.9 microM in all tested regions. Additionally, [Nva2]TRH was shown to compete with [3H]TRH at low affinity TRH-binding sites with similar affinities. It is concluded that the loss of TSH-releasing activity of [Nva2]TRH appears to be due to a drastic reduction in binding affinity to the high affinity TRH receptor subtype. Its analeptic activity, however, may be mediated by low affinity TRH binding sites which are predominantly labelled by [3H]TRH or by yet unidentified mechanisms.

Synthesis and biological activity of 5-fluoroimidazole-TRH

The 5-fluoroimidazole analogue of thyrotropin-releasing hormone, obtained by total synthesis from 5-fluoro-L-histidine, neither binds to rat pituitary cells nor stimulates release of prolactin from them. Lévine-Pinto et. al. reported an agonist, which was generated during presumptive photofluorination of the hormone and which they believed to be the 5-fluoro analogue. In addition to the striking contrast in biological activities, the chemical properties of the agonist differ markedly from those of our peptide and are inconsistent with expectation for the fluoroimidazole moiety. Despite its inactivity in pituitary functions, the authentic 5-fluoro analogue mimics the natural hormone with respect to cardiovascular responses in the central nervous system.

Direct photochemical trifluoromethylation of histidine-containing peptides

Abstract Photochemical trifluoromethylation of the imidazole ring of histidine in the tripeptide Glp-His-Pro-NH 2 (TRH) has been achieved to furnish a mixture of the isomers, 2- and 4(5)-CF3-Im-TRH, which have been separated by reverse-phase HPLC and characterized.

Effect of thyrotropin releasing hormone and some of its histidine analogs on the cardiovascular system and prolactin release in the conscious rat

The cardiovascular and endocrine activity of three analogs of thyrotropin releasing hormone (TRH), 4-nitro-imidazole TRH (4-nitro-TRH), 2-trifluoro-methyl-imidazole TRH (2-TFM-TRH) and 4-trifluoro-methyl-imidazole TRH (4-TFM-TRH), was compared to TRH in conscious rats. Injection of TRH or the three analogs (1 mg/kg or 5 mg/kg) into the arterial line induced increases in mean arterial pressure, pulse pressure and heart rate and raised plasma prolactin (PRL). None of the analogs were more potent than TRH in inducing cardiovascular changes. The 4-TFM-TRH was significantly less potent than the 2-TFM-TRH in increasing blood pressure, while the nitro-TRH was more potent than the 2-TFM-TRH in producing tachycardia. TRH induced a two-fold increase in PRL at the 5 mg/kg dose, while both the fluorinated analogs elicited a 4 to 5 fold increase in PRL at the higher dose. The present results suggest that the receptors for TRH-elicited PRL release differ from TRH-receptors involved in its cardiovascular actions.

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.

Selective cardiorespiratory activity of an iodinated analog of thyrotropin-releasing hormone (TRH)

The biological activity of thyrotropin-releasing hormone (TRH) and its analogs 4(5)-I-Im-TRH and 2,4(5)-I2-Im-TRH was assessed by means of their effects on: 1) the mean arterial pressure (MAP), 2) heart rate (HR), 3) ventilation minute volume (MV), 4) contractility of the rat duodenum, and 5) concentrations of thyrotropin (TSH) or prolactin (PRL) in serum. Also their binding to TRH-receptors in brain homogenates was studied. In urethane-anesthetized rats TRH ICV increased MAP, HR and MV. 4(5)-I-Im-TRH was equally as active as TRH on HR and MV but a significant elevation in MAP was observed only at a dose 100-fold to that of TRH. However, the maximal responses of 4(5)-I-Im-TRH and TRH did not differ. In conscious rats, TRH 1A elevated MAP and HR but 4(5)-I-Im-TRH was active on MAP only. 2,4(5)-I2-Im-TRH was devoid of cardiorespiratory activity. TRH dose-dependently inhibited the contractions of the rat duodenum while the iodinated analogs lacked such an activity. To induce a significant release of TSH several hundred times more of 4(5)-I-Im-TRH and over 1000 times more of 2,4(5)-I2-Im-TRH were needed as compared to TRH. The iodoanalogs elevated PRL levels only at doses 2000-fold higher than those of TRH. The iodoanalogs displaced [3H][3-Me-His2]TRH [( 3H]MeTRH) from its binding sites at concentrations about 1000 times higher than those of TRH. Substitutions of the histidyl moiety of TRH in 4(5)-I-Im-TRH and 2,4(5)-I2-Im-TRH resulted in substantial loss of the endocrine activity. While the di-iodinated analog was practically devoid of any biological activity the monoiodinated analog exerted similar cardiorespiratory activity to that of TRH.

Dissociation of the cardiovascular and prolactin-releasing activities of norvaline2-TRH

The effects of thyrotropin-releasing hormone (TRH) and norvaline2-TRH (Nva2-TRH) on blood pressure, heart rate and plasma prolactin levels in conscious rats have been compared. Systemic injection of TRH or Nva2-TRH (1 mg/kg or 5 mg/kg) produced equipotent increases in plasma prolactin. On the other hand, while TRH significantly increases blood pressure and heart rate, Nva2-TRH was essentially inactive. Thus, two contrasting analogues are now available: 4-NO2-Im-TRH (Neuropeptides, 8, 63, 1986) has full cardiovascular activity and no PRL-releasing activity, while Nva2-TRH has no cardiovascular activity and full PRL-releasing activity of TRH.