Partial characterization of immunoreactive substance P in the rat pituitary gland

Two distinct carboxy-terminus-directed anti-substance P (SP) sera (R-1C and R-6G) were used to characterize immunoreactive SP (I-SP) in acetic acid extracts of anterior pituitary (AP) and posterior pituitary (PP) glands of adult male rats. The tissue concentrations of I-SP measured by R-1C and R-6G were comparable. The contents of I-SP were 600–1150 pg/AP and 25–52 pg/PP. I-luteinizing hormone releasing hormone and I-somatostatin (I-SOM) were undetectable in AP extracts, but PP extracts contained the equivalents of 325–785 pg I-SOM/gland. Serial dilutions of AP and PP extracts produced displacement curves with both SP antisera that were parallel to the respective synthetic SP standard and hypothalamic extract displacement curves. Gel filtrations of AP and PP extracts on a Sephadex G-25 column produced I-SP peaks eluting in the same fractions as synthetic SP and hypothalamic I-SP. However, the AP I-SP profile also revealed a side peak migrating between the void volume and the major I-SP peak. Neither immunoreactive species in the AP extract were eliminated when eluted with 6.0 M guanidine HCl, a strong denaturing agent. $\text{In vitro}$ incubation of paired anterior hemipituitaries for 30 min in the presence of a 56 mM K⁺ concentration resulted in a significant (p<.0001), 25-fold increase in the release of I-SP into the incubation medium above the mean control value. Radiofrequency lesions placed in the median eminence-arcuate region of male rats caused a significant (p<.001) reduction of I-SP in both the AP and PP. These reductions were inversely related to the plasma prolactin values. The elevation in plasma prolactin was taken as an index of completeness of lesions. We conclude that: 1) the rat pituitary contains I-SP as assessed by its immunologic and chromatographic behavior, 2) K⁺ depolarization is a potent stimulator of the release of AP I-SP $\text{in vitro}$, 3) the ME-arcuate region is important for the maintenance of pituitary I-SP levels in the rat.     

Effect of substance P on mucus secretion of isolated submucosal gland from feline trachea

To elucidate how substance P (SP) produces submucosal gland secretion, we examined the effects of SP on the glandular contractile response and 3H-labeled glycoconjugate release in isolated submucosal glands from feline tracheae. SP (10(-12) to 10(-4) M) produced dose-dependent increases in the contractile response, and the maximal tension induced by SP was approximately 70% of the response to methacholine. SP-induced contraction is blocked completely by atropine and augmented by neostigmine. Pretreatment with hemicholinium 3, an acetylcholine synthesis inhibitor, inhibited the contractile response to SP. Pretreatment with tetrodotoxin did not inhibit the contractile response to SP. Capsaicin induced tension of a magnitude similar to that of SP. SP (10(-7) M) produced a significant increase (74% above control) in radiolabeled glycoconjugate release from isolated glands, whereas SP had no significant effects on glycoconjugate release from tracheal explants, probably because of epithelial suppression. Atropine abolished SP-evoked glycoconjugate release in isolated glands. Our findings indicate that 1) SP induces glandular contraction, which is related to the squeezing of mucus in the ducts and secretory tubules, 2) SP stimulates radiolabeled glycoconjugate release in isolated submucosal gland, probably involving mucus synthesis and/or cellular secretion, and 3) these two actions are mediated by a peripheral cholinergic mechanism.     

P物质对大鼠腺垂体培养细胞Camp含量的影响

目的：探讨P物质（SP）作用于大鼠腺垂体培养细胞SP受体（SPR）后是否影响第二信使cAMP的水平。方法：应用RIT法测定细胞内cAMP的含量。结果与结论：SP兴奋腺垂体细胞SPR后的生物学反映至少部分是通过刺激第二信使cAMP的生成来完成的。     

Inappropriate secretion of thyrotropin by the pituitary

Inappropriate secretion of thyrotropin (IST) is characterized by elevated serum free thyroid hormone and unsuppressed thyrotropin (TSH) levels, and results from either a TSH-secreting pituitary tumor (nIST) or a selective resistance to thyroid hormone action (nnIST). Although in most patients TSH levels are definitely high, in a quarter of the cases they are within the 'normal range'. In some of these cases, TSH had an elevated biologic activity and an apparent molecular weight smaller than in normals. The current availability of ultrasensitive TSH immunoradiometric assay, able to distinguish suppressed from unsuppressed TSH levels enables the recognition of the disease. The distinction between nnIST and nIST rests on clinical, neuroradiological, and biochemical criteria, the most useful of which are the alpha-subunit:TSH molar ratio (increased in nIST), and the evaluation of the TSH responses to thyrotropin-releasing hormone and high doses of 3,5,3'-triiodothyronine, both qualitatively normal in nnIST, while absent in nIST. The therapy of choice for patients with nIST is pituitary surgery, followed by irradiation in case of surgical failure. Chronic administration of bromocriptine is effective in a minority of cases. The long-acting somatostatin analogue SMS 201-995 has given promising results in 2 patients. In nnIST, bromocriptine is frequently uneffective, while small doses of 3,5,3'-triiodothyronine or 3,5,3'-triiodothyroacetic acid, a thyroid hormone derivative with a strong inhibitory effect on TSH secretion but poor thyromimetic activity on peripheral tissues, are effective in controlling TSH hypersecretion.     

Age differences in neurokinin A and substance P from the hypothalamus,pituitary, pineal gland,and striatum of the rat. Effect of exogenous melatonin

Previous data showed that aging of the central nervous system (CNS) is associated with widespread changes in tachykinin gene expression. However, there are no data about the possible role of exogenous melatonin in modulating the tachykinergic system during aging. The aim of this work was to analyze the age-dependent changes on neurokinin A (NKA) and substance P (SP) levels in hypothalamus, pituitary, pineal gland and striatum and the role of exogenous melatonin on these changes. We studied female rats at three different ages: 5-month-old (cyclic), 15-month-old (preacyclic) and 25-month-old (acyclic). Hypothalamic tachykinin levels increase when female rats reached acyclicity, this increase was blunted in acyclic-melatonin-treated rats. However, melatonin treatment in young cyclic rats resulted in significantly increased values as compared to controls. Pituitary NKA concentrations did no show age-dependent changes in control rats, however, in both, preacyclic and acyclic-melatonin-treated rats significantly increased values of pituitary NKA were found compared to controls. In the pineal gland, a marked decrease of NKA levels was observed in acyclic-control rats. Melatonin treatment did not alter this decrease. In the striatum, NKA and SP concentrations were significantly reduced in preacyclic- and acyclic-control rats compared to young cyclic rats, melatonin had no effect on striatal tachykinins. Our results indicate that melatonin may regulate tachykinin stores during aging mainly on structures of the neuroendocrine-reproductive axis.     

Localization of thyrotropin (TSH) in the dog pituitary gland

Summary Using the immunoperoxidase technique and antisera to the specific beta () subunits of bovine and rat TSH¹, selective immunocytochemical staining was localized in a specific cell population in the pars distalis of the dog pituitary gland. These TSH cells were found to be positive to aldehyde fuchsin, alcian blue, periodic acid-Schiff (PAS) and aniline blue. With the performic acidalcian blue (pH 0.2) -PAS-orange G procedure these cells stained blue-purple, demonstrating FSH/LH cells (blue or turquoise), ACTH/MSH cells (redpurple) and PRL cells (orange-red). The TSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunocytochemical double staining. In the pars distalis of adult male dogs the TSH cells were mostly shown to be smaller in size and less numerous than in bitches in the anestrous phase of the sexual cycle. Moreover, cytological alterations in the immunoreactive thyrotrophs in the pituitary of male and female dogs generally paralleled the spontaneous changes in thyroid function associated with thyroid atrophy and/or pituitary insufficiency, and thyroid hyperplasia or goiter. In conclusion, because of their specificity and high potency, the antisera to the -subunits of bovine and rat TSH represent an effective tool for the selective immunocytochemical localization of TSH in the dog pituitary. This allows the study of the morphology and function of TSH cells under different physiological, pathological and experimental conditions.Abbreviations for Hormones cited in this Paper ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - TRH TSH Releasing Hormone - CG Chorionic Gonadotropin The authors are grateful to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance     

Effects of dopamine on prolactin secretion and cyclic AMP accumulation in the rat anterior pituitary gland

The effects of dopamine on pituitary prolactin secretion and pituitary cyclic AMP accumulation were studied by using anterior pituitary glands from adult female rats, incubated in vitro. During 2h incubations, significant inhibition of prolactin secretion was achieved at concentrations between 1 and 10nm-dopamine. However, 0.1–1μm-dopamine was required before a significant decrease in pituitary cyclic AMP content was observed. In the presence of 1μm-dopamine, pituitary cyclic AMP content decreased rapidly to reach about 75% of the control value within 20min and there was no further decrease for at least 2h. Incubation with the phosphodiesterase inhibitors theophylline (8mm) or isobutylmethylxanthine (2mm) increased pituitary cyclic AMP concentrations 3- and 6-fold respectively. Dopamine (1μm) had no effect on the cyclic AMP accumulation measured in the presence of theophylline, but inhibited the isobutylmethylxanthine-induced increase by 50%. The dopamine inhibition of prolactin secretion was not affected by either inhibitor. Two derivatives of cyclic AMP (dibutyryl cyclic AMP and 8-bromo cyclic AMP) were unable to block the dopamine (1μm) inhibition of prolactin secretion, although 8-bromo cyclic AMP (2mm) significantly stimulated prolactin secretion and both compounds increased somatotropin (growth hormone) release. Cholera toxin (3μg/ml for 4h) increased pituitary cyclic AMP concentrations 4–5-fold, but had no effect on prolactin secretion. The inhibition of prolactin secretion by dopamine was unaffected by cholera toxin, despite the fact that dopamine had no effect on the raised pituitary cyclic AMP concentration caused by this factor. Dopamine had no significant effect on either basal or stimulated somatotropin secretion under any of the conditions tested. We conclude that the inhibitory effects of dopamine on prolactin secretion are probably not mediated by lowering of cyclic AMP concentration, although modulation of the concentration of this nucleotide in some other circumstances may alter the secretion of the hormone.     

Binding studies of substance P anterior pituitary binding sites: changes in substance P binding sites during the rat estrous cycle

Previous studies have shown that substance P (SP), an undecapeptide widely distributed in the gastrointestinal tract and in the peripheral and central nervous system, is a putative regulatory peptide involved in the control of reproductive function. Specifically, SP inhibited, at the anterior pituitary (AP) level, the stimulatory action of a physiological concentration (10(-8) M) of Gonadotropin Releasing Hormone (GnRH) on the release of the luteinizing hormone (LH). In the present work, we have demonstrated the presence of specific SP binding sites in the AP and related changes in the number of these sites to GnRH receptor number, hypothalamic SP and GnRH content and LH secretion during the rat estrous cycle. High affinity saturable SP binding sites (Kd, 1.5 approximately equal to 10 nM) were demonstrated in AP membranes using [3H]-SP or a novel analog, [125I]-(D-Tyr0, NorLeu11)SP. The binding affinity of SP fragments decreased with progressive removal of amino acid residues from N or C termini of the molecule. Other neuropeptides had low affinity for the SP binding sites. During the rat estrous cycle, SP and GnRH binding capacity of the anterior pituitary were inversely related. At the time of the proestrous LH surge, the AP binding capacity was low for GnRH but high for SP. The highest content of SP in the hypothalamus were recorded during the afternoon of proestrus when hypothalamic GnRH levels were lowest and the preovulatory surge occurred. These studies have established the presence of high affinity specific binding sites for SP in the AP which alter during the estrous cycle in a manner appropriate for mediating the direct inhibitory effects of SP on LH release in vitro.     

Delta-sleep-inducing peptide (DSIP) inhibited CRF-induced ACTH secretion from rat anterior pituitary gland in vitro

Delta-sleep-inducing peptide (DSIP, 10(-9) - 10(-7) M) significantly inhibited the CRF-induced ACTH release from rat anterior pituitary quarters in vitro. 10(-8) M DSIP showed the most prominent inhibition. DSIP (10(-8) M) also inhibited the CRF-activated cAMP levels in anterior pituitary tissue. DSIP did not influence basal ACTH or cAMP levels. Prostaglandin E2 (PGE2)-release from anterior pituitary quarters was not changed by DSIP. From these results, we conclude that DSIP inhibits CRF-induced ACTH release at the pituitary level through the inhibition of the cAMP system in corticotrophs. The involvement of PGE2 in this phenomenon is unlikely.     

Postnatal differentiation of the immunohistochemical expression of aromatase P450 in the rat pituitary gland

At our laboratory, we have recently demonstrated the immunohistochemical expression of aromatase P450 in the pituitary glands of adult rats; this expression was seen to be sex-dependent. In order to determine whether the changes in the expression of the enzyme are related to changes in the gonadal sphere and whether the expression of the enzyme is related to the postnatal differentiation of hypophyseal cytology, in the present work we performed an immunohistochemical study in the rat pituitary gland from birth to old age. The immunohistochemical reaction to aromatase was evident and very generalized at 7 days after birth, with no large differences between the male and female animals. At 14 days the immunohistochemical reaction was decreased in the females, with no changes in the males. At 17 days, aromatase immunoreactivity in the pituitary glands of female rats was very weak whereas the males showed large numbers of reactive cells. These observations were further pronounced at 21 days and 2 months of life. At 24 months, the immunoreactivity found in the pituitary glands of the male rats had almost completely disappeared. Our results show that a postnatal differentiation in the immunohistochemical expression of aromatase occurs; this is tightly linked to sexual activity and is lost in old age. This suggests that hypophyseal aromatase would be related to the mechanisms of action of gonadal steroids on hypophyseal differentiation and secretion.     

Effect of repeated stimulation by thyrotropin-releasing hormone (TRH) on thyrotropin and prolactin secretion in perfused euthyroid and hypothyroid rat pituitary fragments

The previously reported refractoriness of pituitary response to thyrotropin-releasing hormone (TRH) stimuli was investigated here in an in vitro perfusion system using pituitary tissue from euthyroid and hypothyroid rats. Thyroid-stimulating hormone (TSH) and prolactin (PRL) responses to TRH (28 pmol) were significantly greater in hypothyroid tissue compared with euthyroid. Hypothyroid tissue showed a reduction in response to two consecutive stimuli in both TSH and PRL, however the TSH decline in response was more marked than PRL. Euthyroid tissue showed no significant decline in response to TRH. An increase in the dose of TRH (112 pmol), administered to euthyroid tissue, resulted in increased TSH and PRL response, but no decline in response to sequential stimuli was observed. Three consecutive stimuli by TRH (28 pmol) of hypothyroid tissue resulted in a consistent decline in TSH response. The decline in PRL response only reached statistical significance by the third stimulation. Euthyroid and hypothyroid pituitary tissue was subjected to sequential depolarising stimulation with KCl (50 mumol). Euthyroid tissue showed no decline in response in either TSH or PRL. In hypothyroid tissue only, the decline in TSH response reached statistical significance. This decline in TSH response was significantly smaller than the decline in response observed in hypothyroid tissue stimulated with TRH. Refractoriness of hypothyroid pituitary tissue to repeated TRH stimuli is reported here. Our data suggest that the decline in hormonal response cannot be explained solely on the basis of tissue depletion.     

Vitamin A-deficiency and thyrotropin secretion in the rat.

Basal serum TSH concentrations and TRH-induced TSH response were studied in control and in vitamin A-deficient rats at different times between the fifth week on diet (when growth of deficient animals was still normal) and the beginning of the weight plateau (as soon as growth of deficient animals had stopped). In deficient rats the TSH values were always lower than in the control rats. TRH injections (50 ng/100 g b.w.) in anaesthetized animals (amobarbital 1 mg/100 g b.w.) resulted in an approximately 12-fold increase in serum TSH levels within 6 minutes. The TSH levels remained elevated for at least 15 minutes and were similar in control and deficient rats. We hypothesize that the lower basal serum TSH concentrations are the result of a feedback mechanism triggered by an increase of serum free thyroxine (FT4) and free triiodothyronine (FT3).