Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.     

Expression of messenger ribonucleic acids encoding neuropeptide-Y, substance-P, and vasoactive intestinal polypeptide in human pituitary.

The local production of autocrine or paracrine agents in endocrine tissues represents an important level of hormonal regulation. The synthesis of neuropeptide-Y (NPY), substance-P (SP), and vasoactive intestinal peptide (VIP) in the rat anterior pituitary gland has been well demonstrated. We have now studied their expression in human postmortem pituitary tissue. Northern blot analysis of poly(A)+ RNA from whole human pituitaries revealed mRNA encoding the precursors for NPY, SP, and VIP whose hybridization characteristics were indistinguishable from those of the same mRNAs described in previously characterized human tissues. VIP mRNA was detectable in all samples tested, with NPY and preprotachykinin-A mRNA (which encodes SP) detectable in a subset of the pituitaries. The concentration of immunoreactive NPY in whole human pituitary was 3.8 +/- 1.1 pmol/g wet wt in males and 2.9 +/- 0.5 pmol/g wet wt in females (mean +/- SEM; n = 10), that of SP was 3.1 +/- 0.4 pmol/g wet wt in males and 5.2 +/- 1.3 pmol/g wet wt in females (n = 10), and that of VIP was 8.1 +/- 2.9 pmol/g wet wt in males and 5.3 +/- 1.6 pmol/g wet wt in females (n = 10). Size-fractionation of pituitary extracts by gel permeation chromatography revealed single peaks of NPY and VIP-like immunoreactivity in the positions of the standards, while SP-like immunoreactivity mostly eluted in the position of synthetic SP, with two minor immunoreactive peaks eluting earlier. The low levels of NPY, SP, and VIP and their mRNAs in the human pituitary are consistent with peptides having an autocrine/paracrine, rather than endocrine, mode of action.     

Immunoreactive dynorphin in pituitary and brain.

Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peak of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide.     

Anterior pituitary cells from Brattleboro (di/di), Long-Evans and Sprague-Dawley rats contain immunoreactive arginine vasopressin

A radioimmunoassay specific for arginine-vasopressin (AVP) was used to establish the presence of immunoreactive (ir)-AVP in extracts of anterior pituitary glands from Sprague-Dawley (SD) and Long-Evans (LE) rats (3.05 +/- 1.0 and 1.66 +/- 0.9 ng/gland, respectively). Lower levels of ir-AVP (0.56 +/- 0.26 ng/gland) were detected in anterior pituitary gland extracts from rats with hereditary diabetes insipidus (Brattleboro; di/di). The anterior pituitary gland ir-AVP from each rat strain was further characterized by reverse-phase high-performance liquid chromatography (RP-HPLC). In each case the major peak of immunoreactivity co-migrated with synthetic AVP. By peroxidase-antiperoxidase immunocytochemistry, sparsely distributed cells containing ir-AVP were localized in anterior pituitary sections. Levels of ir-AVP in primary cultures of anterior pituitary cells (from SD rats) increased from 52 +/- 5 pg/10(6) cells at 2 days in vitro to 152 +/- 17 pg/10(6) cells at 3 days; during this period 56 +/- 6 pg/ml ir-AVP was secreted into the culture medium. Fewer than 1% of the cells in these cultures were immunostainable for AVP. These data indicate that the anterior pituitary gland of the Brattleboro, Long-Evans and Sprague-Dawley rat contains ir-AVP, and that there is synthesis and secretion of this peptide in primary cultures of anterior pituitary cells in vitro.     

7B2, a new protein secreted by human functionless pituitary tumours, in vitro

A novel pituitary protein, 7B2, of approximately 180 amino acids has been suggested to colocalise with LH in the pituitary gonadotropes. Increased secretion of LH is known to occur in functionless pituitary tumours. We have therefore measured 7B2 immunoreactive equivalents in the 24-h medium of explant pituitary cultures prepared from 17 functionless, 20 somatotropic, 16 PRL secreting and 8 corticotropic adenomas. A synthetic fragment corresponding to amino acids 23-39 of 7B2 was used to raise antisera (rabbits), prepare radiolabel (chloramine T iodination) and also serve as the assay standard. 7B2-immunoreactive equivalents in the medium from the functionless tumours was 517 +/- 149 pmol/l, significantly higher than that of the somatotropic tumours (248 +/- 90 pmol/l, P less than 0.05), prolactinomas (108 +/- 37 pmol/l, P less than 0.001) and corticotropin producing adenomas (107 +/- 77 pmol/l, P less than 0.001) (one-way analysis of variance). Gel permeation chromatography of medium obtained from functionless tumours revealed two immunoreactive 7B2 peaks one eluting at a coefficient of 0.28 corresponding to that of normal human pituitary extract and another eluting at a coefficient of 0.59. Gel chromatography profiles of medium obtained from somatotropic tumours contained similar immunoreactive 7B2 peaks (elution coefficient 0.28 and 0.57). These findings demonstrate that 7B2-like material is secreted by pituitary adenomas in explant culture with the highest level from functionless tumour cultures.     

A dynorphin-like opioid in the central nervous system of an amphibian.

We have provided evidence for the existence of a biologically active opioid in toad (Bufo marinus) brain that is immunoreactive with antiserum raised against dynorphin (1-13). Compared with porcine dynorphin, this opioid is similar in apparent molecular weight on the basis of gel permeation chromatography and is more hydrophobic on the basis of high-performance liquid chromatography. After purification, its opioid biological activity was demonstrated on the guinea pig ileum myenteric plexus-longitudinal muscle preparation. It was found to be less potent, and to have a similar sensitivity to antagonism by naloxone, in comparison with porcine dynorphin. Because it is immunoreactive with antiserum specific for porcine dynorphin, it probably has considerable sequence homology. Generally, the tissue distribution of immunoreactive dynorphin in the toad is similar to that in the rat, with highest concentrations in the neurointermediate lobe of the pituitary. However, the anterior lobe of the toad pituitary contains considerably lower concentrations than are found in the rat anterior lobe. There appear to be three size classes of immunoreactive dynorphin in toad neural tissue, each with apparent molecular weight below 12,000, similar to the size classes of immunoreactive dynorphin found in pig and rat. However, in toad spinal cord (and possibly in brain) there is immunoreactive dynorphin of greater apparent molecular weight, which has not been reported in mammalian tissue. The contribution of each molecular size to the total immunoreactivity varies from tissue to tissue and is different from that observed in the rat.     

Growth hormone specific stimulation of mitosis by size-fractionated serum from patients with growth hormone insufficiency: a study on the rat lymphoma cell line, Nb2

The aim was to investigate the bioactivity of a high-molecular weight human growth hormone, identified following molecular sieve chromatography of serum. Nine patients with pituitary disease and GH insufficiency were studied. All patients had non-detectable levels of immunoreactive GH, less than 0.2 micrograms/l, in diurnal serum profiles. GH bioactivity was determined before and after size-fractionation of serum. The bioassay is based on the finding that a rat lymphoma cell line, Nb2, proliferates in the presence of lactogens. GH and PRL immunoreactivities were measured by radioimmunoassays. Pronounced GH immunoreactivity was found in fractions of sera from 7 out of the 9 patients and of 2 of 4 control sera, particularly in fractions corresponding to the elution volume of high-molecular weight proteins (greater than 160 kD). PRL immunoreactivity was only detected in fractions corresponding to the elution volume of monomeric PRL. Unfractionated serum had a dose-dependent mitogenic effect on the Nb2 cells. GH-antibodies could not inhibit this effect. Fractions of serum obtained from the patients stimulated Nb2 cell division as well. The mitogenic effect of serum fractions could be inhibited by GH-antibodies. Thus, high-molecular weight GH circulating in patients with GH insufficiency were shown to exert a GH-specific bioactivity in vitro after size-fractionation.     

A novel pituitary protein (7B2)-like immunoreactivity is secreted by a rat phaeochromocytoma cell line (PC12)

High concentrations of a novel pituitary protein (7B2) have been shown to be present in the PC12 rat phaeochromocytoma cell line by radioimmunoassay. 7B2-like immunoreactivity (IR-7B2) was released from PC12 cells into the incubation medium in response to stimulation by a depolarizing concentration of K+, and this K+-evoked release was inhibited by Co2+. The major IR-7B2 in PC12 cell and medium appeared to be identical to that in porcine pituitary gland as judged by both gel permeation chromatography and by reverse-phase high performance liquid chromatography (HPLC). Gel permeation chromatography of extracts of cell and medium revealed two IR-7B2 peaks, the earlier eluting at a elution coefficient (Kav) of 0.30 and the later at a Kav of 0.54. In medium, over 90% of the IR-7B2 eluted as the earlier peak. Fractionation of extracts of cell and medium on reverse-phase HPLC showed three main IR-7B2 peaks eluting at 43, 44.5 and 46% acetonitrile/water with 0.1% trifluoroacetic acid. The findings suggest that IR-7B2 might be released by calcium-mediated exocytosis.     

Corticotrophin-releasing factor-like immunoreactivity and bioactivity of human fetal and adult hypothalami

Corticotrophin releasing factor-like immunoreactivity (CRF-LI) and bioactivity, and arginine vasopressin-like immunoreactivity (AVP-LI) have been measured in extracts of human fetal and adult hypothalamic tissue and their development with the gestational age of the fetuses (12-27 weeks) studied. CRF-LI was measured by a radioimmunoassay developed for ovine corticotrophin-releasing factor (oCRF-41). Corticotrophin-releasing factor bioactivity was measured in a rat isolated anterior pituitary cell perfusion system. CRF-LI and bioactivity and AVP-LI were all detectable in fetal hypothalamic extracts from 12 to 13 weeks of gestational age. CRF-LI was also present in human fetal pituitary glands from 12 weeks of gestational age. The concentration of CRF-LI in the fetal hypothalamic extracts (9.2 +/- 11.4 ng/g, mean +/- S.E.M., n = 33) showed no significant correlation with the gestational age of the fetuses. However the concentration of AVP-LI (25.0-36.8 ng/g, n = 17) did show a positive correlation (r = 0.508, P less than 0.05) with gestational age, as did the concentration of CRF bioactivity (471.3-556.3 ng ACTH released/g tissue, n = 13, r = 0.725, P less than 0.01). The CRF bioactivity of all fetal hypothalamic extracts was potentiated by the addition of synthetic human (h)AVP, but the bioactivity of the adult hypothalamic extracts was not, presumably because of the higher levels of AVP-LI already present in the adult extracts. Pretreatment of tissue extracts with antisera to oCRF-41 and/or hAVP reduced the CRF bioactivity of all hypothalamic extracts. Sephadex chromatography of fractions which co-eluted with synthetic oCRF-41 or hAVP contained CRF bioactivity and this bioactivity was potentiated when synthetic hAVP or oCRF-41, respectively, were added to the fractions. However, a larger molecular weight form of CRF-LI (8000-10 000 daltons), which was observed only in fetuses of 20 weeks of gestational age or less, did not contain any significant CRF bioactivity.     

Axons containing a prolactin-like peptide project into the perivascular layer of the median eminence: an immunocytochemical light and electron microscope study in adult and infant rats

A light and electron microscopic immunocytochemical study was undertaken to explore the fine structural organization of prolactin-immunoreactive axons in the rat median eminence. In adult intact males and females and in hypophysectomized females, light microscopic immunocytochemical labeling of the mediobasal hypothalamus revealed a marked concentration of prolactin-like immunoreactive fibers in the perivascular layer throughout the median eminence and the hypophysial stalk. At the electron microscopic level, immunostaining was associated with typical neurosecretory axons located either in the palisade layer where they displayed numerous contacts with tanycyte processes, or in the perivascular layer where they frequently contacted the perivascular space. Within the labeled axonal profiles, immunostaining was essentially located on secretory granules, 90-120 nm in diameter, whereas the microvesicles accumulated in some perivascular profiles constantly remained unlabeled. These data strongly suggest that most prolactin-immunoreactive axons of the median eminence release their content into the hypophysial portal vessels. In 1-day-old infant rats, intensely prolactin-like immunoreactive fibers were similarly localized in the most external layer of the median eminence, in which, contrary to adult animals, very slight if any tyrosine-hydroxylase-immunoreactive fibers were detected. Since earlier studies have provided evidence for a nondopaminergic prolactin-release-inhibiting factor in the hypothalamus of infant rats, and for an inhibitory effect of prolactin on pituitary mammotrophs, we propose that hypothalamic prolactin may contribute, as an additional prolactin-release-inhibiting factor, to the multifactorial control of pituitary mammotrophs.     

Regulation by thyroid hormone of the concentration of substance P in the rat anterior pituitary

The content of immunoreactive substance P (iSP) in the male rat anterior pituitary was measured after thyroidectomy and excess T4 administration. Baseline values for iSP content (mean +/- SE, 400 +/- 37 fmol/mg protein) increased progressively after thyroidectomy (4 days, 893 +/- 100; 6 days, 1321 +/- 242; 14 days, 1897 +/- 509). Administration of pharmacological doses of T4 (50 micrograms daily) for 2 and 14 days significantly decreased anterior pituitary iSP content (2 days, 196 +/- 30; 14 days 138 +/- 12). Thyroid status did not affect iSP content in the hypothalamus, caudate nucleus, amygdala, or brainstem. Partial chemical characterization of SP immunoreactivity in the anterior pituitary was obtained by gel permeation chromatography on Sephadex G-25, high pressure liquid chromatography, and the use of two antisera in RIAs, one directed against the amino-terminus and one directed against the carboxyl-terminus of the peptide. SP in the anterior pituitary was readily releasable in vitro by 44 mM potassium chloride in a calcium-dependent manner. The present study demonstrates that the concentration of iSP in the rat anterior pituitary is affected by the thyroid status of the animal and supports the probability that thyroid hormones participate in the regulation of the synthesis and/or release of iSP from the anterior pituitary.     

Immunoreactive dynorphin in mammalian spinal cord and dorsal root ganglia.

The distribution of immunoreactive dynorphin (ir-dynorphin) has been determined in dorsal and ventral aspects of spinal cord and in dorsal root ganglia of rabbit and rat. Concentrations are highest in dorsal root, with intermediate levels in ventral cord and low levels in dorsal root ganglia of both species. Levels of ir-dynorphin are relatively uniform over examined segments (vertebrae C2-S3) of rabbit spinal cord and dorsal root ganglia. Gel permeation chromatography of extracts from rabbit dorsal and ventral spinal cord and dorsal root ganglia revealed at least three immunoreactive components of differing molecular size in all three structures. Multiple unilateral or bilateral dorsal rhizotomy (vertebrae C5-T1) in rat did not affect levels of ir-dynorphin in spinal cord. As reported [Goldstein, A. & Ghazarossian, V. E. (1980) Proc. Natl. Acad. Sci. USA 77, 6207-6210], midthoracic spinal transection was without effect. Within the spinal cord, the neuropeptide appears, most probably, to be contained in short-axoned neurons. We surmise that this potent opioid peptide may participate in the processing of sensory information in spinal cord.     

The presence, characterisation and synthesis of neuromedin B in the human pituitary gland.

Neuromedin B is a 10-amino-acid mammalian peptide of the bombesin family. We have used a specific radioimmunoassay and Northern blot hybridisation to investigate the possible synthesis of neuromedin-B-like immunoreactivity in the human pituitary gland. The concentration of immunoreactive neuromedin B in whole human pituitary was 15.2 +/- 4.2 pmol/g wet weight in males and 12.8 +/- 2.7 pmol/g wet weight in females (mean +/- SEM, n = 10). In pituitary tumour extracts, neuromedin B immunoreactivity was 9.1 +/- 1.7 pmol/g wet weight (mean +/- SEM, n = 14) in inactive tumours, 18.4 +/- 6.9 pmol/g wet weight (mean +/- SEM, n = 4) in somatotrophs and 10.4 +/- 2.7 pmol/g wet weight (mean +/- SEM, n = 2) in prolactinomas, with no apparent significant difference between the groups. Gel permeation chromatography of pituitary extracts revealed two immunoreactive peaks, the major one of which corresponded in position to that of neuromedin B-32 and a later minor peak to the position of the neuromedin B-10 standard. On fast protein liquid chromatography, neuromedin-B-like immunoreactivity again eluted in two peaks, a minor peak corresponding to the synthetic neuromedin B standard, and a major more hydrophobic peak which was the big neuromedin B form. Northern blot analysis of poly(A)+RNA from human pituitaries revealed the presence of a hybridising band of between 750 and 850 base pairs. These results suggest that neuromedin B is synthesised in the human pituitary gland where it may be of importance in the regulation of pituitary function. Furthermore, the adenomatous condition is not associated with abnormal levels of this peptide.     

Identification of multiple low molecular weight placental prolactin-like proteins produced by rat trophoblast cells

Rat trophoblast tissue was found to synthesize a number of low molecular weight proteins possessing prolactin-like characteristics. There appear to be at least three proteins that cross-react with antisera to pituitary prolactin. Two of the proteins had a molecular weight of 25,000, similar to ovine pituitary prolactin, and isoelectric points of 6.8 and 7.0. The third immunoreactive protein had a lower molecular weight (23,500), similar in size to human placental lactogen, and a slightly more acidic isoelectric point of 6.75. The molecular weight variants cross-reacted with an antipeptide serum that was generated to a synthetic peptide representing amino acids 150 to 164 of rat placental lactogen-2 (PL-2). Based on this analysis, we consider these proteins to be related to PL-2. Analysis of trophoblast proteins by gel-filtration chromatography resulted in the identification of another trophoblast prolactin. This material eluted earlier than PL-2-related proteins on a gel-filtration column, possessed prolactin-like activity (determined by competition with ovine pituitary prolactin for rabbit mammary gland or rat liver prolactin receptors) but showed limited cross-reactivity with either the antiserum to pituitary prolactin or the antiserum to the PL-2 peptide. We have thus identified multiple low molecular weight trophoblast prolactins, possessing different biochemical and immunological characteristics.     

The mechanism of action of cysteamine in depleting prolactin immunoreactivity

The thiol reagent cysteamine (CSH) depletes anterior pituitary cells of immunoreactive PRL both in vivo and in vitro. We examined the hypothesis that CSH affects either the solubility or immunoreactivity of PRL through a mechanism involving thiol-disulfide exchange. Adult female rats were treated with either CSH (300 mg/kg, sc) or an equimolar dose of ethanolamine as a control. Anterior pituitary glands were extracted in 0.1 M sodium borate buffer, pH 9.0. Treatment of pituitary extracts with beta-mercaptoethanol (BME) destroys the immunoreactivity of PRL. However, extraction in the presence of reduced glutathione or CSH of pituitaries of rats treated with CSH restores immunoreactive PRL to control levels. Extracts were also subjected to polyacrylamide gel electrophoresis (PAGE). On gels of pituitary extracts of CSH-treated rats, the band that comigrates with purified PRL is diminished compared to that in ethanolamine-treated controls. This is found regardless of whether the borate extracts are treated with BME. However, extraction of the pituitaries in sodium dodecyl sulfate-containing buffer followed by chemical reduction with BME restores the PRL band. Therefore, CSH acts on PRL through a thiol-related mechanism to yield a product that is poorly soluble in aqueous buffer at pH 9 and is poorly immunoreactive. Dispersed anterior pituitary cells in tissue culture were incubated with L-[35S]methionine to radiolabel newly synthesized peptides. These cultures were incubated in the presence of either CSH or ethanolamine. PAGE followed by autoradiography confirmed the above results obtained in vivo. Also, extracts of CSH-treated cultures were subjected to gel permeation chromatography. As determined by PAGE, at least some of the radiolabeled PRL can be recovered from void volume fractions by reduction with BME, indicating that CSH induces the formation, through disulfide exchange, of a high mol wt form of PRL, possibly PRL oligomers.     

Heterogeneity of human growth hormone and prolactin secreted in vitro: Immunoassay and radioreceptor assay correlations.

Gel filtration of sera and of in vitro pituitary incubation media from a normal subject and from subjects with pituitary tumors or physiologically elevated growth hormone (hGH) and/or prolactin (hPRL) levels, has been performed. Heterogeneous immunoreactive forms of both hormones with comparable elution profiles in sera and in in vitro incubation media were identified. In each instance the most retarded component ('little hPRL' and 'little hGH') migrated identically with the respective radioiodinated pituitary standard and constituted the major component identified. Several components of intermediate mobility were identified and characterized as 'big hPRL', 'big hGH,' and 'big big hGH'. In addition, a void volume immunoreactive peak was often identified. No interconversion was demonstrated on refiltration of prolactin tumor sera or normal pituitary incubation media. Radioreceptor activity utilizing pregnant rabbit liver membranes for hGH and rabbit mammary membranes for hPRL indicates comparable immunologic and receptor activity in all forms identified following gel filtration of prolactin tumor sera (for hPRL) and normal pituitary incubation media (for hGH). Only for the larger species of HGH, identified by gel filtration of clinical grade hGH, was diminished receptor activity demonstrated. These data suggest that the human pituitary synthesizes and secretes protein hormones of different molecular size according to gel filtration elution volume profiles but with comparable receptor and immunologic assay reactivity. These results support but do not establish the proposal that pituitary hormones are also secreted as prohormones.     

Identification of latent tissue kallikrein, prolactin and growth hormone secretion in GH3 pituitary cells using modified radioimmunoassays

Our studies demonstrate that rat anterior pituitary cells (GH3) are capable of synthesizing and secreting tissue kallikrein together with prolactin and growth hormone. The secretion of prolactin and growth hormone in GH3 cells was measured by two newly developed sensitive radioimmunoassays (RIA), using the polyethylene glycol separation technique. In the direct radioimmunoassay for rat tissue kallikrein, using a polyclonal antiserum which recognizes both active and prokallikrein, the GH3 kallikrein displays parallelism with standard curves of rat urinary kallikrein. The production of immunoreactive kallikrein, prolactin, and growth hormone is time-dependent, and the levels after a 72 h incubation in serum-free media are approximately 12.2 +/- 4.4 ng, 272.2 +/- 33.0 ng, and 475.6 +/- 4.8 ng per 10(6) cells per ml (mean +/- SD, n = 3), respectively. In Western blot analyses, a specific monoclonal antibody to tissue kallikrein (V4D11) identifies GH3-secreted kallikrein as a approximately 39,000 Da protein, slightly larger than approximately 38,000 Da kallikreins of submandibular gland, mouse anterior pituitary cells (AtT 20) or rodent neuroblastoma X glioma hybrid cells (NG108). Kallikrein mRNA in GH3 cells was identified in Northern blot analyses, using a tissue kallikrein cDNA probe. In a RIA using a kallikrein monoclonal antibody (V1C3) recognizing only active kallikrein, kallikrein could not be detected in the media incubated up to 48 h with GH3 cells. However, after trypsin treatment, a time-dependent increase of immunoreactive kallikrein (using monoclonal antibody V1C3), Tos-Arg-OMe esterase, and kinin-releasing activities can be measured in the conditioned media. The activated esterase activity was inhibited by aprotinin and by affinity-purified kallikrein monoclonal antibody (V4D11) in a dose-dependent manner. The data indicated that rat anterior pituitary GH3 cells secrete latent tissue kallikrein, which can be converted to active kallikrein by trypsin. These hormonally responsive cells co-synthesize kallikrein with prolactin and growth hormone and provide a model system for studying the regulation of kallikrein gene expression.     

Rat brain natriuretic peptide--tissue distribution and molecular form

Using an antiserum against the ring structure of rat brain natriuretic peptide (rat BNP), we have established a specific radioimmunoassay (RIA) for rat BNP and elucidated its tissue distribution and molecular form. Rat BNP-like immunoreactivity (-LI) was detected in the highest amount in cardiac extracts (574.0 +/- 138.8 pmol/g in the atrium and 4.3 +/- 1.1 pmol/g in the ventricle). The secretory rate of rat BNP-LI from the perfused whole heart was 50.0 +/- 1.9 fmol/min, about 60% of which was maintained even after atrial removal. We also detected rat BNP-LI throughout the spinal cord (134-175 fmol/g), although no detectable amount was present (less than 100 fmol/g) in other tissues including the brain. High performance-gel permeation chromatography and reverse phase-high performance liquid chromatography coupled with the RIA revealed that rat BNP with 45 amino acids is a major storage form as well as a secretory form of rat BNP-LI in the heart. The major component in the spinal cord was also rat BNP. These findings indicate that the tissue distribution and the processing pattern of rat BNP are different from those of atrial natriuretic peptide and porcine BNP, thereby suggesting the presence of complicated diversity of the natriuretic peptide system.     

Concentrations of immunoreactive thyrotropin-releasing hormone in spinal cord of patients with amyotrophic lateral sclerosis

Concentrations of immunoreactive thyrotropin-releasing hormone (ir-TRH) were measured by specific radioimmunoassay in the spinal cord of six patients with amyotrophic lateral sclerosis (ALS) and seven with non-neurological diseases. Ir-TRH concentrations were the highest in the anterior horn, compared with other areas of the spinal cord, both in non-neurological diseases and ALS. Ir-TRH concentrations in the anterior horn of ALS were significantly lower than in non-neurological diseases, but were the same in both groups in other parts of the spinal cord (e.g. posterior horn, frontal part, lateral and central part, posterior part). Ir-TRH concentrations in rat spinal cords were stable for up to seven hours when spinal cord was stored after death at 4 degrees C or 22 degrees C. An elution profile of methanol-extracted human spinal cord on Sephandex G-10 column was identical to that of synthetic TRH. The cell population in the anterior horn in ALS was decreased markedly. The findings suggest that TRH is present in the human spinal cord and its decreased concentrations in the anterior horn of ALS may be due to a decrease in the cell population.