Cardiac Thyrotropin-releasing Hormone Inhibition Improves Ventricular Function and Reduces Hypertrophy and Fibrosis After Myocardial Infarction in Rats.

BACKGROUND: Cardiac thyrotropin-releasing hormone (TRH) is a tripeptide with still unknown functions. We demonstrated that the left ventricle (LV) TRH system is hyperactivated in spontaneously hypertensive rats and its inhibition prevented cardiac hypertrophy and fibrosis. Therefore, we evaluated whether in vivo cardiac TRH inhibition could improve myocardial function and attenuate ventricular remodeling in a rat model of myocardial infarction (MI). METHODS AND RESULTS: In Wistar rats, MI was induced by a permanent left anterior descending coronary artery ligation. A coronary injection of a specific small interfering RNA against TRH was applied simultaneously. The control group received a scrambled small interfering RNA. Cardiac remodeling variables were evaluated one week later. In MI rats, TRH inhibition decreased LV end-diastolic (1.049 ± 0.102 mL vs 1.339 ± 0.102 mL, P < .05), and end-systolic volumes (0.282 ± 0.043 mL vs 0.515 ± 0.037 mL, P < .001), and increased LV ejection fraction (71.89 ± 2.80% vs 65.69 ± 2.85%, P < .05). Although both MI groups presented similar infarct size, small interfering RNA against TRH treatment attenuated the cardiac hypertrophy index and myocardial interstitial collagen deposition in the peri-infarct myocardium. These effects were accompanied by attenuation in the rise of transforming growth factor-ß, collagen I, and collagen III, as well as the fetal genes (atrial natriuretic peptide, B-type natriuretic peptide, and beta myosin heavy chain) expression in the peri-infarct region. In addition, the expression of Hif1α and vascular endothelial growth factor significantly increased compared with all groups. CONCLUSIONS: Cardiac TRH inhibition improves LV systolic function and attenuates ventricular remodeling after MI. These novel findings support the idea that TRH inhibition may serve as a new therapeutic strategy against the progression of heart failure.

[ß-Glu2]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain.

Selective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu2]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [ß-Glu2]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [ß-Glu2]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [ß-Glu2]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.

Thyrotropin-releasing hormone: stimulation of colonic activity following intracerebroventricular administration.

Intraventricularly administered thyrotropin-releasing hormone in rabbits elicited an increase in intraluminal pressure changes, a response commonly associated with muscular activity of the colon. The response appears to be central in origin with peripheral expression relying primarily on cholinergic receptors.

Inhibition of gastric acid secretion in rats by intracerebral injection of corticotropin-releasing factor.

Intracisternal injection of ovine corticotropin-releasing factor (CRF) into the pylorus-ligated rat or the rat with gastric fistula resulted in a dose-dependent inhibition of gastric secretion stimulated with pentagastrin or thyrotropin-releasing hormone. When injected into the lateral hypothalamus--but not when injected into the cerebral cortex--CRF suppressed pentagastrin-stimulated acid secretion. The inhibitory effect of CRF was blocked by vagotomy and adrenalectomy but not by hypophysectomy or naloxone treatment. These results indicate that CRF acts within the brain to inhibit gastric acid secretion through vagal and adrenal mechanisms and not through hypophysiotropic effects.

Thyroid hormone inhibition of the prolactin response to thyrotropin-releasing hormone.

The influence of serum triiodothyronine (T(3)) and thyroxine (T(4)) concentrations on the release of prolactin in man was studied by determining the prolactin response to synthetic thyrotropin-releasing hormone (TRH) in hypothyroid and hyperthyroid patients before and after correction of their serum thyroid hormone abnormalities. The maximum increment in serum prolactin above the basal level (maximum Delta prolactin) was used as the index of response to TRH. In 12 patients with primary hypothyroidism, the maximum Delta prolactin in response to TRH fell from 100.5+/-29.1 ng/ml (mean +/-SEM) before treatment to 36.1+/-6.0 ng/ml (P < 0.01) during the 4th wk of treatment with 30 mug T(3) + 120 mug T(4) daily. The mean serum T(3) level increased from 57+/-8 to 138+/-10 ng/100 ml, and the mean serum T(4) level increased from 3.0+/-0.4 to 7.2+/-0.4 mug/100 ml during this treatment. In eight normal subjects the maximum Deltaprolactin in response to TRH was not significantly different during the 4th wk of treatment with 30 mug T(3) + 120 mug T(4) daily from the response before treatment. In 10 patients with hyperthyroidism, the maximum Deltaprolactin in response to TRH increased from 14.2+/-2.9 ng/ml before treatment to 46.9+/-6.7 ng/ml (P < 0.001) during antithyroid treatment. The mean serum T(3) level fell from 313+/-47 to 90+/-8 ng/100 ml, and the mean serum T(4) level fell from 20.8+/-2.5 to 6.8+/-0.6 mug/100 ml during this treatment. These results show that changes from normal serum levels of T(3) and T(4) are associated with changes in prolactin responses to TRH; subnormal serum levels of T(3) and T(4) increase TRH-induced prolactin release, whereas substantially higher than normal serum levels of T(3) and T(4) inhibit this release.

Regulation of body temperature by thyrotropin-releasing hormone in neonatal chicks.

To understand thermal regulation of neonatal chicks, the contribution of thyrotropin-releasing hormone (TRH), a key regulator of the hypothalamus-pituitary-thyroid axis, was investigated. Intracerebroventricular (i.c.v.) injection of TRH (5 and 20 microg) increased body temperature, but did not change plasma T3 and T4 concentrations. Intraperitoneal (i.p.) injection of triiodothyronine (T3) and thyroxine (T4) did not influence body temperature. Thereafter, the relationships between TRH and the hypothalamus-pituitary-adrenal axis and sympathetic nervous system were further investigated. Central TRH stimulated both corticosterone and epinephrine release. The i.c.v. injection of a corticotropin-releasing factor receptor antagonist attenuated the change in body temperature and corticosterone concentration caused by TRH, but did not influence plasma T3 and T4 concentrations. The i.p. injection of epinephrine did not induce hyperthermia. Therefore, the thermoregulatory response to TRH may differ in neonatal stages being dependent upon the stimulation of the hypothalamus-pituitary-adrenal axis rather than the hypothalamus-pituitary-thyroid axis.

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Mechanism of thyroid hormone inhibition of thyrotropin-releasing hormone action.

The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.

The response of newborn sheep to TRH with and without somatostatin.

Hormone responses to a bolus injection of thyrotropin releasing hormone (TRH) were studied in 8 newborn lambs between 6 and 19 hours of age. The effect of a bolus injection and 45 min infusion of somatostatin (SRIF) on these responses was studied in 2 other animals. Serial measurements of serum TSH, prolactin, triiodothyronine (T3) and thyroxine (T4) were conducted for 2 to 6 h in all animals. Mean baseline T4 and T3 concentrations were 12.6 microng/dl and 221 ng/dl, respectively, both significantly higher than values in fetal or adult animals. These high values were due to the events of parturition. In spite of the high baseline T4 and T3 levels, there were rapid and significant increases in both TSH and prolactin concentrations in response to TRH alone. The TSH response evoked further increments in serum T3 and T4 concentrations observed at 30 min and 60 min, respectively, both subsequently increasing progressively through 6 h. During the 45 min period of SRIF infusion, the TSH T4 and T3 responses to the zero time TRH injection were minimal. However, after discontinuing SRIF, late increases in TSH, T4 and T3 were observed. The results indicate that the hyperiodothyroninemia characteristic of the newborn period does not block the response to exogenous TRH, whereas the inhibitory effect of exogenous SRIF is observed in the newborn as in the adult. The increased endogenous TRH secretion presumably responsible for the neonatal TSH surge may be overriding the negative feedback effect of thyroid hormones.

Tetracaine, propranolol and trifluoperazine inhibit thyrotropin releasing hormone-induced prolactin secretion from GH3 cells by displacing membrane calcium: further evidence that TRH acts to mobilize cellular calcium.

TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells. Tetracaine (1 mM), propranolol (1 mM), and trifluoperazine (0.03 mM) inhibited basal and TRH-stimulated prolactin release, decreased cellular 45Ca2+ content and decreased cell-associated CTC fluorescence. Most importantly, these agents abolished the decrease in CTC fluorescence induced by TRH. These data suggest that tetracaine, propranolol and trifluoperazine displace membrane Ca2+ in intact GH3 cells and offer further evidence that TRH acts to mobilize cellular Ca2+ from a membrane-bound pool(s) during stimulation of GH3 cells.

Evidence for a thyrotropin-releasing hormone inhibitor in the pineal gland.

Extracts of bovine pineal glands were filtered through Sephadex G-25 and tested for their effect on TRH-induced TSH release in cell cultures of dispersed rat pituitary glands. Highly significant anti-TRH activity was found in material which was retarded on G-25. This material was purified sequentially on Sephadex G-10, Sephadex LH-20, and high pressure reverse phase liquid chromatography columns. The ability of partially purified material to inhibit TRH-induced TSH release by dispersed pituitary cells and to displace [3H]TRH from pituitary tumor cell membranes was interpreted as evidence for a TRH inhibitory factor (TRH-IF) in bovine pineal glands. Evidence for binding to TRH receptors included parallel competition displacement curves with synthetic TRH and data showing that excess TRH could overcome both the inhibition of TRH-induced TSH release and the inhibition of [3H]TRH binding by TRH-IF. Copurification of anti-TRH bioactivity (as tested in dispersed pituitary cell cultures) and anti-TRH-binding activity (as tested in membrane preparations) suggests that the inhibitory activity in pineal extracts results from the binding of a TRH antagonist to receptors. Reports by others of PRL release-inhibiting activity in pineal extracts were confirmed. PRL release-inhibiting activity copurified with TRH-IF.

U-73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing hormone effects in GH3 rat pituitary cells.

TRH increases cytosolic-free calcium ([Ca2+]i) by activating phospholipase C(PL-C), which induces phosphoinositol hydrolysis, leading to Ca2+ mobilization from inositol trisphosphate (IP3) sensitive stores, and by increasing Ca2+ influx. Increases in [Ca2+]i stimulate PRL secretion. We investigated the effects of U-73122, an aminosteroid inhibitor of PL-C dependent processes, on TRH-stimulated second messenger pathways and on PRL secretion in GH3 rat pituitary cells. [Ca2+]i was monitored by Indo-1 fluorescence, and IP3 and metabolites separated on ion exchange columns. In Ca(2+)-free buffer, [Ca2+]i was 96 +/- 6 nM and increased to 323 +/- 23 nM (P less than 0.001) after TRH (100 nM). U-73122 dose dependently inhibited the TRH effect (IC50 = 967 nM; complete inhibition at 3-5 microM). Subsequent addition of monensin (100 microM) increased [Ca2+]i from 107 +/- 4 to 142 +/- 4 nM (P < 0.001), confirming our previous findings of a non-TRH regulated Ca2+ pool in GH3 cells. Pretreatment (15 sec) with U-73122 partly inhibited the TRH effect on [Ca2+]i; complete suppression occurred with 70 sec of pretreatment. An inactive analog (U-73343) had no inhibitory effect at 5 microM. U-73122 acted noncompetitively, as the mean maximum velocity (expressed as percent increase in [Ca2+]i after TRH) was reduced from 225 to 91 while the Michaelis-Menten constant for TRH was unchanged (15.4 vs. 13.8 nM, n = 3). Of note, U-73122, at 3-5 microM, increased basal [Ca2+]i from 109 +/- 5 to 120 +/- 5 nM (P less than 0.001). In 1.3 mM Ca2+ buffer containing nifedipine (1 microM) and verapamil (50 microM), similar effects of U-73122 (5 microM) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 +/- 12%, 148 +/- 23%, and 167 +/- 39% of control, 30 sec after TRH (100 nM); these responses were prevented by 1 microM U-73122. At 5 microM, U-73122 also significantly increased IP3 levels. TRH (100 nM) increased 4-h PRL secretion from 16.3 +/- 1.4 to 27.6 +/- 3.2 ng/well (P less than 0.05). U-73122 (5 microM) increased basal PRL secretion to 35.9 +/- 3.2 ng/well (P less than 0.05), but abolished the TRH effect. In contrast, U-73343 (with Ca2+ channel blockers) did not inhibit the TRH effect on PRL (control: 24.3 +/- 2.1; TRH: 51.0 +/- 6.3 ng/well).(ABSTRACT TRUNCATED AT 400 WORDS)

Sphingosine interacts directly with the receptor complex to inhibit thyrotropin-releasing hormone binding.

Sphingosine inhibition of [3H] [N3-Me-His] TRH (MeTRH) binding, previously shown to be independent of its effects on protein kinase-C, has been further characterized in GH3 cell membranes and in a partially purified, digitonin-solubilized receptor preparation. In membranes, as in intact cells, sphingosine inhibited [3H]MeTRH binding by decreasing receptor affinity, but, in contrast to its effect in intact cells, did not affect the number of available binding sites. The inhibition of binding was linear up to 75 microM sphingosine (in the presence of 100 microM BSA at 0.1 mg membrane protein/ml), yielding an apparent Ki of 51 microM. Since GTP decreases the affinity for MeTRH binding in GH3 cell membranes, we studied interactions between GTP and sphingosine. While the effects of low concentrations of GTP gamma S and sphingosine were additive, sphingosine inhibition of MeTRH binding surpassed and was not affected by the addition of maximally inhibitory concentrations of GTP gamma S. Also, sphingosine (75 microM) did not affect the ability of a maximally effective dose of TRH to stimulate the low Km GTPase (vehicle, +35 +/- 5%; sphingosine, +32 +/- 10%); there was a 25% decrease in total GTPase activity in the presence of sphingosine. MeTRH binding to digitonin-solubilized receptors, which had properties similar to those described previously by others, including no effect of GTP on binding, was inhibited by sphingosine. In solubilized receptors, as in membranes, sphingosine caused a decrease in apparent affinity without changes in the number of binding sites. These data suggest that sphingosine interacts directly with the TRH receptor [or an associated factor(s) in the receptor complex] to decrease affinity by a mechanism that does not involve uncoupling of G-proteins.

Benzodiazepines inhibit thyrotropin (TSH)-releasing hormone-induced TSH and growth hormone release from perifused rat pituitaries.

The perifusion technique was used to investigate the action of diazepam (DZ), a benzodiazepine molecule known to compete for TRH receptor binding in rat pituitary, on TRH-induced TSH and GH release. The release kinetics for the two hormones from quartered pituitaries were measured in response to a 6-min pulse of TRH (10 nM), without or with DZ addition for a period of 30 min before and during the TRH pulse, plus an additional 15-min period. The dynamic patterns of TSH and GH release in response to TRH were characterized by a rapid increase in hormone release, declining slowly over the next 20 min. The rate of release represented 2.98 +/- 0.02 (+/- SE) and 1.75 +/- 0.06 times the corresponding basal level for TSH and GH, respectively, when evaluated over the first 15 min of the response to TRH. Addition of increasing doses of DZ suppressed the stimulatory effect of TRH in a dose-related manner, with an ID50 of 3 nM for both TSH and GH. The maximal effect of DZ was obtained with a concentration of 10 nM for both hormones. Ro 15-1788 (100 nM), a selective antagonist of the central type of benzodiazepine-binding sites (added to the perifusion system 30 min before DZ and then during the whole period of DZ perifusion), completely abolished (P less than 0.01) the inhibitory effect of DZ (10 nM) on the TRH-induced TSH and GH responses. When added alone before the TRH pulse, Ro 15-1788 had no effect on the TSH response to TRH. In contrast, PK 11,195 (100 nM), a selective antagonist of the nonneuronal benzodiazepine-binding sites, was unable to abolish the inhibitory action of DZ on TRH-stimulated TSH release. In addition, the effects of four other types of benzodiazepine (flurazepam, chlordiazepoxide, midazolam, and medazepam), all tested at a 10-nM concentration, corroborated these findings. Furthermore, DZ inhibition of the TSH response was nullified by picrotoxin (1 microM), but not by bicuculline (1 microM), two gamma-aminobutyric acid antagonists that had no effect, by themselves, on this response. For comparison, the effect of DZ (10 nM) was also tested on the release of GH in response to human GH-releasing factor-(1-44)-NH2 (10 nM) and was found to be ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a dual effect of gamma-aminobutyric acid on thyrotropin (TSH)-releasing hormone-induced TSH release from perifused rat pituitaries.

The effects of gamma-aminobutyric acid (GABA) on the spontaneous and TRH-induced TSH release were investigated in vitro on perifused rat pituitaries. The dynamic pattern of TSH release was measured in response to a 6-min pulse of TRH (10 nM) with or without GABA addition. GABA had no effect on spontaneous TSH release but exhibited a dual effect on TSH-stimulated release according to the dose (as calculated by the induced-basal ratio): a potentiation of the TSH response to TRH at the lowest concentrations tested (less than or equal to 10 nM) and an inhibition for GABA concentrations equal or higher than 100 nM. The GABA potentiation was mimicked by muscimol (10 microM) and isoguvacine (10 nM) but not by baclofen (1 microM). Bicucullin (1 microM) or picrotoxin (1 microM) added 15 min before GABA was unable to reverse the GABA potentiation of the TSH response, although SR 95103 (1 and 10 microM), a specific GABA A antagonist, partially or totally antagonized this response. Diazepam (7 nM) was able to potentiate the TSH response by 216% when GABA was added to the system at a concentration (60 nM) which does not modify by itself the TSH response. The inhibitory effect of GABA (100 nM) was completely abolished by bicucullin (1 microM), by picrotoxin (1 microM), and by SR 95103 (1 microM). Picrotoxin not only blocked the inhibitory action of GABA but significantly (P less than 0.05) potentiated the TSH response to TRH. Our data suggest a dual GABA-ergic control of TRH-stimulated TSH release directly on the pituitary, probably mediated by two different kinds of GABA receptors: a GABA A receptor site mediating the inhibitory effect and a nonclassical GABA A receptor site of higher affinity for its stimulatory action.

Prolactin inhibition by p-tyramine in the male rat: site of action.

In a previous report, a consistent hypoprolactinemic effect of p-tyramine was observed in male rats under several experimental conditions in vivo. In the present experiments the action of p-tyramine on PRL release in vitro, or after challenge with different hyperprolactinemic drugs (serotonin, morphine, and TRH) was tested. Furthermore the participation of octopamine, a metabolite of tyramine, was evaluated with regard to the hypoprolactinemic action of the amine. P-Tyramine inhibited PRL release from hemipituitaries incubated in vitro at doses of 10(-4) and 10(-6) M (inhibition to 31% and 59% of control values, respectively). When tested for its ability to displace [3H]spiperone binding in vitro to a crude fraction of anterior pituitary membranes it was found that it did not compete with the D2 receptor labeled by [3H]spiperone, even at the concentration of 10(-4) M. P-Tyramine (40 mg/kg) antagonized the elevation of serum PRL levels by morphine, serotonin, and TRH. On the other hand, octopamine, which is formed from tyramine, also inhibited high PRL values found after stress, though the effective dose was higher than that of tyramine. Pretreatment with diethyldithiocarbanic acid, which inhibits conversion of p-tyramine to octopamine, did not modify the effect of tyramine in stress. The present results indicate that tyramine can inhibit PRL release due to certain drugs, by acting directly at the pituitary level. It does not displace [3H]spiperone binding from anterior pituitary membranes, and octopamine which lowers PRL release itself, cannot account for the effect of tyramine.

Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells.

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.

Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+.

Salmon calcitonin (sCT)-like peptide is present in the central nervous system and pituitary gland of the rat, and this peptide inhibits basal and TRH-stimulated PRL release from cultured rat anterior pituitary (AP) cells. The present studies were designed to examine further the inhibitory actions of sCT on basal and TRH-stimulated PRL release and investigated 1) the temporal dynamics of the responses, 2) the effects of sCT on PRL release induced by other secretogogues, and particularly those acting via elevations of cytosolic Ca2+, and 3) the selectivity of sCT action on basal and stimulated AP hormone release. The inhibition of basal PRL release by sCT (0.1-10 nM) was dose-dependent and was characterized by a rapid onset with a gradual recovery to normal rates of release after the period of sCT inhibition. The inhibitory effect of sCT on basal PRL release was reversed by treatment with either the Ca2+ ionophore A23187 or with the phorbol ester, phorbol myristate acetate (PMA). sCT infusion did not affect the basal release of GH, TSH, FSH, or LH by perifused AP cells. When administered in short pulses, TRH, at concentrations from 1-100 nM, elicited a dose-dependent increase in PRL release. When coadministered with short 10 nM TRH, sCT (1-100 nM) inhibited TRH-induced PRL release in a dose-dependent manner, with a maximal inhibition of 78% at a concentration of 10 nM, and an ED50 concentration of approximately 3 nM. During longer (30 min) pulses of TRH (100 nM), PRL release increased sharply over 4-fold within 2 min, followed within 12 min by a rapid decline to a level 1.5-2-fold higher than basal, and this level was maintained for the remainder of the stimulation period. sCT pretreatment inhibited the overall PRL response to TRH. In contrast to its inhibition of TRH-induced PRL release, sCT failed to prevent the stimulation of PRL release by either ionophore A23187, PMA, vasoactive intestinal peptide, or forskolin. In addition, sCT failed to block TRH-induced TSH release or GnRH-induced LH release.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.

Deletion of the Nhlh2 transcription factor decreases the levels of the anorexigenic peptides alpha melanocyte-stimulating hormone and thyrotropin-releasing hormone and implicates prohormone convertases I and II in obesity.

Body weight is controlled by the activation of signal transduction pathways in both the brain and peripheral tissues. Interestingly, although many hypothalamic neuropeptides and receptors have been implicated in the regulation of body weight, the transcriptional and posttranscriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Our laboratory studies a mouse in which targeted deletion of the neuronal basic helix-loop-helix (bHLH) transcription factor, nescient helix-loop-helix 2 protein (Nhlh2), results in adult-onset obesity. The aim of this work was to use the phenotype of the Nhlh2 knockout mouse and the expression pattern of Nhlh2 to identify genes that are regulated by this transcription factor. In this article, we show that Nhlh2 is expressed throughout the adult hypothalamus. Using dual-label in situ hybridization, we demonstrate that, in the arcuate nucleus of the adult hypothalamus (ARC), Nhlh2 expression can be found in rostral proopiomelanocortin (POMC) neurons, whereas in the paraventricular nucleus (PVN), Nhlh2 is expressed in TRH neurons. In addition, we find that hypothalamic POMC-derived alphaMSH in the ARC and TRH in the PVN are regulated posttranscriptionally via Nhlh2-mediated control of prohormone convertase I and II mRNA levels. This is the first report in which regulation of body weight is linked to the action of a neuronal bHLH transcription factor on prohormone convertase mRNA levels. Furthermore, this work supports a direct role for transcriptional control of neuropeptide processing enzymes in the etiology of adult-onset obesity.