Effect of substance P on thyrotropin secretion from the pituitary gland in the rat

The role of substance P (SP) on thyrotropin (TSH) secretion was investigated in ovariectomized (OVX) female, estrogen-primed OVX, and normal male rats. Third ventricular administration of SP induced a significant increase in plasma TSH levels when compared to control animals in E-primed OVX rats (p less than 0.001). The plasma TSH levels increased in a dose-related manner and reached maximum levels at 10 min after injection. In contrast, intraventricularly injected SP failed to alter plasma TSH levels in both OVX rats and normal male rats. Intravenous administration of SP dramatically stimulated TSH release in E-primed OVX rats (p less than 0.001), whereas SP had no effect on the release of TSH when injected in OVX rats and normal male rats. To investigate any direct action of SP on TSH release from the anterior pituitary gland, synthetic SP was incubated with dispersed anterior pituitary cells harvested from E-primed OVX rats and normal male rats. SP, in the dose range between 10(-8) M and 10(-6) M, failed to alter the release of TSH into the culture medium in vitro. These findings indicate that SP has a stimulatory role in the control of TSH release by an action on the hypothalamus but only in estrogen-primed rats.     

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.     

Ethanol-related changes in substance P in the hypothalamus and anterior pituitary.

The effect of the administration of a rabbit anti-substance P serum (ASPS) was studied in rats receiving an acute injection of ethanol. ASPS lowered serum prolactin levels and reduced the hyperprolactinemia induced by ethanol. ASPS also decreased LH serum levels in both saline- and ethanol-treated rats. The effect of ethanol on the concentration of substance P-like immunoreactivity (SP-LI) in the mediobasal hypothalamus and the anterior pituitary gland was also investigated. Ethanol reduced SP-LI in the mediobasal hypothalamus but increased it in the anterior pituitary gland. The presence of ethanol (50 mM) did not affect the K(+)-evoked release of SP-LI from either mediobasal hypothalamus or anterior pituitary gland, though it increased the SP-LI concentration remaining in this gland. These results indicate that ethanol increases the content of SP-LI in the anterior pituitary gland and suggest that substance P may be involved in the prolactin release induced by the acute administration of ethanol.     

Tentative immunohistochemical demonstration of melatonin in the rat pineal gland

Summary In the present study an attempt was made to demonstrate melatonin in the rat pineal gland by means of immunohistochemistry. The anti-body used was raised against 5-methoxy-N-acetyltryptophan which is chemically similar to melatonin. Specific fluorescence was demonstrable only in pineals from rats killed during the night, when melatonin formation is high. It was restricted to parenchymal cells lying in a marginal zone of the organ. These results are discussed in relation to a subdivision of the pineal parenchyma into cortical and medullary areas.Supported by a grant of the Deutsche Forschungsgemeinschaft (VO 135/4) within the Schwerpunktprogramm Neuroendokrinologie     

Determination of rat pineal gland melatonin content by a radioimmunoassay.

Melatonin content in individual rat pineal glands was measured by radioimmunoassay (RIA). The RIA used can very reliably detect as little as 50 pg of melatonin. The various precursors, analogues, and the metabolite of melatonin (6-hydroxymelatonin) which were tested for cross-reactivity were not recognized by the antibody. The effects on melatonin levels in rat pineal glands following the administration of L-tryptophan, 5-hydroxy-L-tryptophan, serotonin, N-acetylserotonin, melatonin and pargyline are also presented.     

Osmotic regulation of substance P and neurokinin A peptide content and substance P binding sites in distinct hypothalamic nuclei of the rat.

Quantitative receptor autoradiography using Bolton-Hunter iodinated substance P (SP) was used to localize specific sites in the rat hypothalamus. The amount of SP and neurokinin A (NkA) in extracts from discrete areas of the hypothalamus was measured using specific radioimmunoassays. A high density of SP binding sites was observed in the perimeter of the magnocellular paraventricular and supraoptic nuclei, while the magnocellular nuclei themselves possessed a low receptor density. In control animals, the number of SP binding sites was also low in the arcuate nucleus and the median eminence. Substance P and NkA peptide concentrations were highest in the paraventricular nucleus (PVN), decreasing in the following order: arcuate nucleus (Arc) greater than median eminence (ME) greater than supraoptic nucleus (SON) greater than subfornical organ (SFO). In animals given 340 mmol/l NaCl instead of tap water to drink for 12 days, significant increases in the number of SP binding sites occurred in the medial parvocellular subdivision of the PVN, periamygdaloid cortex, medial preoptic nucleus, Arc, and ME, but other hypothalamic areas were unaffected. In saline-treated animals, significant increases in SP and NkA peptide concentrations were observed in the ME, while in the SFO only the concentration of NkA increased significantly. In the SON, substance P and neurokinin A levels were doubled, whereas in the PVN and Arc no changes in peptide levels were observed. Chronic osmotic stimulation is associated with lowered circulating levels of adrenocorticotropin releasing hormone (ACTH), and the present data further substantiate the hypothesis that hypothalamic tachykinin-containing neuronal terminals are centrally involved in the inhibition of anterior pituitary ACTH release observed during chronic osmotic stimulation.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.     

Sex-related differences in the nuclear population of postpubertal rat pineal gland. A quantitative study.

Male and female parenchymal pineal cell types have been studied throughout postpubertal development to determine the existence of sex-related differences on a time basis. Six age groups (2, 3, 4, 8, 15 and 24 months) of eight rats (4 males and 4 females) were used in this study. Nuclei of both parenchymal pineal cell types were counted in 5 areas of 26.377 microns 2 per pineal gland on semithin sections. Nonparametric statistics of our results (Mann-Whitney U-test and Kruskal-Wallis H-test) demonstrated significant differences between male and female pinealocytes through the stages studied. In all age groups, the number of nuclei per unit area was larger in female rats. Pineoglial cells did not show significant sex-related differences.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Immunohistochemical localization of substance P in the pineal gland of the domestic pig

An immunohistochemical study of the pig pineal gland was carried out using polyclonal rabbit antiserum raised against substance P (SP). The pineal glands were taken from the newborn, 21-day- and 7-month-old female pigs. Immunoreactive nerve fibers were observed in the pineal gland as well as in the posterior commissure and habenular areas. The bundles of SP-immunoreactive fibers were also seen in the subependymal layer of the pineal tissue. The single SP-immunoreactive nerve fibers and few small bundles of nerve fibers were located with equal density throughout the pineal gland, in the connective tissue septa and in the parenchyma. SP-immunoreactive cell bodies were observed in the medial habenular nucleus. The obtained results point to this nucleus as one of the central sources of SP innervation in the pig pineal gland. The study did not show any differences in the distribution and the density of SP-immunoreactive nerve fibers between newborn, 21-day- and 7 month-old pigs.     

Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.     

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K and neuropeptide gamma.

Modulation of the hypothalamo-pituitary-gonadal axis and the pineal gland by neurokinin A, neuropeptide K, and neuropeptide gamma. PEPTIDES 1999. Neurokinin A (NKA), neuropeptide K (NPK) and neuropeptide gamma (NPG) are members of the family of tachykinins, and act preferentially on NK-2 tachykinin receptors. These peptides are widely distributed and are potent stimulators of smooth muscle contraction, especially in the respiratory and gastrointestinal tract. They also induce vasodilatation and plasma extravasation. Through their effects on the vascular tone, they are also potential regulators of the blood flow and therefore of the function of many organs and tissues. Tachykinins have been demonstrated to influence the secretory activity of endocrine cells, and they may have a physiological role as regulators of endocrine functions. A number of reports have indicated that NPK, NKA and NPG act on the hypothalamo-pituitary gonadal axis to regulate functions related to reproduction. Therefore, we thought that, at this point, it was important to review the available evidence suggesting the role of these tachykinins on reproductive functions by effects exerted at 3 different levels of regulation: the hypothalamus, the anterior pituitary and the gonads. These 3 tachykinin peptides were reported to have effects on reproductive functions, acting on the control of the secretion of gonadotropin and prolactin at the level of the hypothalamo-pituitary axis, and on the steroid secretion by the testes and the ovaries. Acting on the hypothalamus, tachykinins, mainly NPK, were reported to inhibit LH secretion, but this effect is dependent on the presence of gonadal steroids. On the anterior pituitary gland, however, tachykinins were shown to stimulate LH and prolactin secretion, and this effect is also dependent on the presence of gonadal steroids. Tachykinin concentrations in the hypothalamus and pituitary are regulated by steroid hormones. In the hypothalamus, estrogens and testosterone increase tachykinin concentration. In the anterior pituitary gland, estradiol and thyroid hormones markedly depress tachykinin concentrations. Ovariectomy and exposure to short photoperiods significantly increase anterior pituitary tachykinins in the Siberian hamster. In the pineal gland, SP and NK-1 receptors are present and, more recently, the presence of NKA and probably also NPK was demonstrated. Castration and steroid replacement modified the content of tachykinins in the pineal gland. The removal of the superior cervical ganglia was followed by an increase in NKA content in the pineal gland. These results suggest that gonadal steroids may influence tachykinins in the pineal gland. In the gonads, tachykinins stimulated the secretory activity of Sertoli cells, but inhibited testosterone secretion by Leydig cells. There are very few reports on the role of tachykinins in the ovary, but some of them indicated that these peptides are present in some of the ovarian structures, and they may affect the secretion of ovarian steroids. Thus, NKA, NPK and NPG appear to have a modulatory role, mainly acting as paracrine factors, on the hypothalamo-pituitary-gonadal axis.     

Effect of lithium on the melatonin production in the pineal gland of viscacha

Melatonin is synthesized primarily in the pineal gland. Lithium affects the circadian rhythms that may explain its therapeutic effectiveness in the treatment of bipolar disorder. The objective of this study was to investigate the effect of lithium on the biochemical parameters involved in melatonin synthesis in the pineal gland of viscacha. Viscachas were daily intraperitoneally injected with lithium chloride or saline solution for one month. Pineal mRNAs encoding β₁-adrenoceptor and arylalkylamine-N-acetyltransferase enzyme (AA-NAT) were studied by in situ hybridization. Pineal melatonin concentrations were determined by radioimmunoassay, and AA-NAT and hydroxyindol-O-methyltransferase (HIOMT) activities were investigated by radiometric assays. The only parameters that decreased significantly were the expression of AA-NAT mRNA and pineal melatonin levels. Our data suggest that lithium treatment may decrease melatonin synthesis in the viscacha pineal gland by a complex mechanism that involves currently unknown events that are beyond a decrease in the expression of AA-NAT enzyme.     

Distinct binding sites for substance P and neurokinin A (substance K): co-transmitters in rat brain

The distribution of binding sites in rat brain for iodinated neurokinin A and iodinated substance P were compared using autoradiography. Distinct patterns of binding for the two iodinated tachykinins were noted. Binding sites for iodinated neurokinin A were noted in the olfactory bulb, cortex, supraoptic n., paraventricular n., certain amygdaloid n., hippocampus, medial habenula, interpeduncular n., n. of the tractus solitarius, and dorsal horn of the spinal cord. This pattern was in contrast to low levels of binding of iodinated substance P to the cortex, supraoptic n., paraventricular n., and the interpeduncular n., but substantial density of binding sites in numerous other regions.     

Arg3]substance P and neurokinin A from chicken small intestine

Using antisera specific for NH2-terminal and COOH-terminal regions of substance P and for the COOH-terminal region of neurokinin A, peptides with tachykinin-like immunoreactivity were isolated from extracts of chicken small intestine. The peptide Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 differs from human substance P by substitution of the lysyl residue by an arginyl residue at position 3. Synthetic [Arg3]substance P showed identical chromatographic and immunochemical properties to chicken substance P and was equipotent with substance P in contracting the guinea pig ileum. A second peptide His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 isolated from the extracts is identical to human neurokinin A. A third peptide was immunoreactive towards the COOH-terminally directed anti-serum to substance P only but was not characterized structurally in this study.