Susan E. Leeman

The stability and metabolism of intravenously administered neurotensin in the rat

The clearance and metabolism of synthetic and tritiated (3H) neurotensin (NT) were studied following its intravenous injection in a pharmacologic dose (500 pmol/kg) into anesthesized rats. Immunoreactive NT (iNT), measured in a radioimmunoassay (RIA) with use of a carboxyl-(C)-terminal directed antiserum, displayed an apparent half-life (t 1/2) of 0.55 min, while that measured by an amino-(N)-terminal directed antiserum had a t 1/2 of 5 min. The radiolabel from injected 3H-NT (3H on Tyr3,11) had a t 1/2 of 6.5 min. High-pressure liquid chromatography of extracts of plasma obtained from the circulation 0.5-3 min after injection of NT and 3H-NT showed the presence of NT and the generation mainly of the fragments NT1-8, NT1-11, and NT9-13, as well as free 3H-labeled tyrosine. The apparent half-lives of intravenously injected synthetic NT1-8, NT1-11 and NT1-12 measured with the N-terminal RIA were 9, 5 and 5 min, respectively, while that for NT9-13 was less than 0.5 min. These results indicate that exogenously injected NT is rapidly metabolized to form N-terminal fragments which are cleared more slowly than NT. These findings suggest that use of N-terminal antisera to detect the release of endogenous NT into the circulation is likely to yield measurements of the fragments NT1-8 and NT1-11 which thus far have been found to be biologically inactive.

Studies on the role of catecholamines in the regulation of the developmental pattern of hypothalamic aromatase

Experiments were conducted to study the regulation of the developmental pattern of aromatase in the forebrain of the perinatal rat. Two experimental designs were used: aromatase measured in primary cultures of fetal hypothalamic cells and in cell-free preparations of forebrain tissue excised at varying ages. In cultured cells, aromatase decreased logarithmically at a slow rate (t1/2 = 7.8 days). Norepinephrine caused a pronounced dose (4 x 10(-6) M) and time-dependent (2-6 days) drop in aromatase without affecting the levels of 5 alpha-reductase or substance P. In isolated tissue, aromatase activity was compared with the concentrations of norepinephrine and dopamine in the forebrain of males vs females at different perinatal ages and in discrete forebrain areas at postnatal day 4. In no case was a sex difference in catecholamines seen. An overall developmental decline in aromatase was associated with developmental increases in catecholamine levels. Acute treatment with the beta-agonist, isoproterenol, had no effect on brain aromatase activity.

Neurotensin and substance P

Publisher Summary This chapter focuses on the peptides, neurotensin (NT) and substance P (SP), and addresses the literature that relates to the potential roles of NT and substance P as hormones or paracrine agents in the gastrointestinal tract. NT is a tridecapeptide with the amino acid sequence: Glp-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu. NT-containing cells appear first in the duodenum in the embryo although in the adult they are largely found in the distal small intestine. Neurotensin has been found in a subpopulation of epithelial cells, called N cells, scattered throughout the intestinal epithelium. The chapter discusses the presence of NT in nerve fibers, tissue determination by radioimmunoassay (RIA) and high-performance liquid chromatographic (HPLC), metabolism of NT released from the intestine, and biological effects of NT on gastrointestinal tissues. SP is an undecapeptide with the amino acid sequence: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. This sequence is clearly related to those of a number of nonmammalian peptides and to those of the mammalian decapeptides neurokinin α and neurokinin β.

Regulation of adenohypophyseal messenger RNAs in female rats by age, hypothyroidism, estradiol and neonatal androgenization ☆

Hormonal regulation of adenohypophyseal messenger ribonucleic acids (mRNAs) encoding preprotachykinin (PPT), prolactin (PRL) and thyrotropin beta subunit (TSH beta) was examined in juvenile and pubertal female rats. Hypothyroidism, initiated on day 2 (d2) or 22 (d22) of life, increased PPT and TSH beta mRNAs but decreased PRL mRNA 17 days later. Exogenous estradiol given for 3 days reduced PPT mRNA in pubertal (d38) but not juvenile (d18) euthyroid females; conversely, estradiol increased PRL mRNA on d18 but not d38. In hypothyroid females however, estradiol decreased PPT and TSH beta mRNAs at both ages and increased PRL mRNA in pubertal but not juvenile females. Thus, regulation of adenohypophyseal mRNAs by estradiol varies with age and thyroid status. In previous studies, adenohypophyseal tachykinins increased in male, but not female rats at puberty. This sex difference was not reproduced here by neonatal androgenization of females, suggesting that it is not mediated by hypothalamic sexual differentiation. However, PRL mRNA increased in androgenized females; this increase was prevented by ovariectomy, suggesting its medication by estradiol.

Neurotensin gene expression in the rat preoptic area. Implications for the regulation of reproduction.

In diverse mammalian species, neurotensin (NT) is especially abundant in the preoptic area/hypothalamusl and may play an important role in neuroendocrine function. NT released into hypophysial portal blood, principally by neurons in the arcuate nucleus,* may directly influence the secretion of prolactin, growth hormone, and thyroidstimulating hormone from cells of the anterior pituitary gland3-6 (see also McCann & Vijayan, this volume). In addition, NT neurons in hypothalamic regions appear to regulate anterior pituitary function indirectly by influencing the activity of other neurosecretory cells. Support for this view initially came from studies in which central administration of NT produced effects on anterior pituitary function opposite from those caused by its systemic administration.4 We have been investigating the possibility that NT neurons in the preoptic area regulate the hypothalamic-pituitarygonadal axis of the female rat. The gene encoding NT and neuromedin N (NT/N) has been isolated and s e q u e n ~ e d ~ ~ ~ (see Dobner et a l . , this volume), permitting investigation of its expression in rat brain,9 and this review will focus on our recent data indicating that NT/N gene expression in the rostra1 preoptic area of the rat is sexually differentiated and, in the female, estrogen dependent and cyclical.

Isolation and identification of a polypeptide in the HSP 70 family that binds substance P

During the course of an attempt to purify the substance P (SP) receptor from horse salivary glands by substance P-affinity chromatography, a polypeptide of Mr = 78,000 was isolated. The first fifteen amino acid residues at the amino terminus were determined and, unexpectedly, were found to be identical with the amino terminus of a glucose-regulated protein (GRP) of the same molecular weight, a protein that has been identified as a member of the heat shock protein family. This finding raises the intriguing possibility that SP may interact in vivo with GRPs and other members of the heat shock protein family and play a role in modulating their biological activities.

Inhibition of neurotensin release by a cyclic hexapeptide analog of somatostatin

Studies were performed to determine whether the cyclic hexapeptide analog of somatostatin, cyclo(N-Me-Ala-Tyr-D-Trp-Lys-Val-Phe) II, could alter circulating levels of neurotensin (NT) and inhibit the release of NT from small intestine following the intraluminal perfusion of lipid and ETOH. The small intestine of anesthetized rats was perfused with 0.9% NaCl, 1mM ETOH, 100 mM ETOH or 1 mM oleic acid with and without the intravenous infusion of the somatostatin analog. Plasma samples collected from the superior mesenteric vein were extracted, chromatographed on HPLC and assayed with both C-terminal and N-terminal antisera to NT. The basal circulating levels of chromatographically and immunochemically identified NT observed during the perfusion of the small intestine with 0.9% NaCl were significantly lower (p less than 0.01) during the IV infusion of the somatostatin analog as compared to animals infused IV with saline. The 2-3 fold increase in plasma levels of NT observed with the intestinal perfusion of oleic acid and ETOH did not occur in animals simultaneously infused IV with the somatostatin analog. The somatostatin analog was also effective in decreasing the basal levels of NT metabolite NT(1-8) as well as inhibiting the increase in this metabolite that accompanies the stimulated release of NT.

IMMUNOCYTOCHEMICAL STUDY OF NEUROTENSIN LOCALIZATION IN THE MONKEY SPINAL CORD

Radioimmunoassayable neurotensin (NT) has been found in the rat spinal cord and immunocytochemical studies at the light microscopic level have shown that immunoreactive neurotensin (iNT) is present almost exclusively in the dorsal horn of the spinal cord both within numerous fibers in laminae 1 and 11 5s4 and in cell bodies located primarily in the region adjoining laminae I1 and III.s. At the electron microscopic level in the rat, axon terminals that contain iNT have been observed to participate in axodendritic synapses.:. 9 Previous studies in our laboratory have examined the distribution and morphologic features of substance P! and enkephalin neuronal elements in the monkey spinal cord, where differences have been observed in the pattern of distribution of these peptides among rat, cat, and monkey. In the present study, we investigated the localization of iNT in the monkey spinal cord.

Chromatographic and Immunochemical Characterization of Neurotensin in Cat Adrenal Gland and Its Release during Splanchnic Nerve Stimulation

A subpopulation of norepinephrine-containing cells in the cat adrenal medulla contain neurotensin (NT) immunoreactive material. Using high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera we have demonstrated that electrical stimulation of the splanchnic nerve releases NT and its metabolites NT(1-8) and NT(1-12) from cat adrenal gland into the circulation. Blood samples from anesthetized cats were simultaneously collected from the adrenolumbar vein and femoral artery over a 10-min period prior to and during splanchnic nerve stimulation. Trains of stimuli (15 Hz/10V) were applied at 30-s intervals over 10 min. Plasma samples were extracted, run on HPLC and column fractions assayed with C- und N-terminal directed antisera. During splanchnic nerve stimulation unchanged. Concomitant with the increase in NT was a two- to threefold increase in the levels of NT(1-8) and NT(1-12). The chromatographic profiles of extracted adrenal glands showed a major peak of immunoreactive material with the same retention time as NT (18.6 +/- 3.0 pmol/g weight wet of tissue) as well as a small peaks on NT(1-8) (0.96 +/- 0.18 pmol/g) and NT(1-12) (2.05 +/- 0.1 pmol/g). HPLC analysis of a tryptic digest of the NT-like material gave 1.0 equivalent of NT(1-8) and NT(9-13). These results are consistent with the presence of NT in adrenal tissue and its release and metabolism during stimulation of the splanchnic nerve.

ELEVATION OF NEUROTENSIN IN THE CENTRAL REGULATION OF LUTEINIZING HORMONE RELEASE

Neurons immunoreactive to gonadotropin releasing hormone (GRH) are localized to the medial preoptic septa1 region of the rat hypothalamus, an area known to be essential for the regulation of ovulation and luteinizing hormone (LH) surge. Consequently, any bioactive substances found in this region of the medial preoptic nucleus are possible candidates as neurotransmitters or neuromodulators affecting GRH release. Since high concentrations of radioimmunoassayable neurotensin (NT) (144 t 23 fmol/mg protein) are found in this region, this peptide and other bioactive substances were microinjected into this area and circulating levels of LH measured. Experiments were performed on Dial-urethane anesthetized rats three weeks after ovariectomy, since in preliminary experiments, we have found that this anesthetic effectively depresses the pulsatile pattern of plasma LH normally observed in these animals. Fifty nanoliters of saline containing 20 pmoles of NT was injected into the rostral portion of the medial preoptic nucleus. The precise location of the injection site is given by the following coordinates taken from the stereotaxic atlas of Pellegrino ul.: AP = 8.12 mm from vertical zero plane, L = 2.0 mm from the midsagittal suture and angled medially at 8 from the perpendicular, V = 7.0 mm below dura. Thirty minutes after the stereotaxic microinjection, plasma LH levels were increased up to 200 2 28% above control (p < .01, actual concentrations were 441 ? 42 to 97s * 128 ng/ml) and remained significantly elevated over the next 30 rnin (see Fro. 1). A similar stimulatory response was observed following the microinjection of norepinephrine (0.3 nmoles in SO nl) into the same site (FIG. I ) . In contrast, saline, substance P , Leu-enkephalin, and GRH microinjected in the rostral medial preoptic nucleus had no effect (FIG. I). The small, discrete nature of this NT-sensitive region is demonstrated by the observation that injections of NT 0.4 mm rostral or caudal to AP 8.2 no longer produced an effect on LH levels. In order to provide additional evidence for a physiological role for NT in this region, the presence of specific binding sites for NT in tissue homogenates of dissected medial preoptic areas was examined using I Iz5--NT. Increasing concentrations of I -NT ( S O Ci/m mol) were incubated in Tris-acetate (50 mM, pH = 7.2) for 30 minutes at 4°C with washed ( 4 ~ ) tissue homogenates of 10 medial preoptic areas (% mg of total tissue wet weight). 200 pl aliquots of the incubation mixture (containing 62 p g of total protein) were filtered under reduced pressure through GFC glass-fiber filters. A specific component of binding was observed when the nonspecific binding, defined as the binding measured in the presence of 1 pM unlabeled NT, was substracted from the total binding. Scatchard analysis of this specific binding suggested the presence of two binding sites for NT with Kds of 4.2 nM and 29.0 nM and site concentrations of 0.11 pmolesimg and 0.68 pmolesimg, respectively. Thus, the presence of both NT and specific binding sites of this peptide in the medial preoptic area, together with the observed functional effect of microinjec-