Rat Anterior Pituitary Homogenates Research Articles

Bioactive atrial natriuretic factor-like peptides in rat anterior pituitary

The presence of biologically active atrial natriuretic factor (ANF)-like peptides was demonstrated in rat anterior pituitary. ANF-like immunoreactivity was detected in rat anterior pituitary by specific radioimmunoassay and was extracted from rat anterior pituitary homogenates by heat-activated Vycor glass beads; extracts were purified by reverse-phase high performance liquid chromatography. Two peaks containing ANF immunoreactive material were obtained. The first peak was eluted from the C18 mu Bondapak column at a position similar to the 28-amino acid carboxy terminal peptide (Ser99-Tyr126)-ANF of prohormone. The second peak had the same pattern of elution as the 126-amino acid prohormone, (Asn1-Tyr126)-ANF. The biological activity of the smaller molecular weight peptide (28 amino acid) was assessed by its inhibitory effect on 10(-8) M ACTH-stimulated aldosterone secretion in rat zona glomerulosa cell suspension. This ANF-like material also displaced I125-labelled ANF from rat glomerular receptors with a potency similar to synthetic (Arg101-Tyr126)-ANF. Immunocytochemical localization revealed a distribution of ANF-stained cells similar in pattern and location to that of gonadotrophs. These results suggest the existence of biologically active ANF-like peptides and ANF prohormone within the anterior pituitary. However, their role remains to be elucidated.

Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes.

Six vasoactive intestinal peptide (VIP) analogs inhibited [125I]iodo-VIP and [125I]iodo-helodermin binding to high-affinity VIP receptors in rat hepatic membranes. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP greater than [D-Phe2]VIP greater than [D-Arg2]VIP, with the latter two peptides acting as partial agonists only. All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [D-Ser2]VIP, [D-His1]VIP, [D-Arg2]VIP and [D-Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [D-Phe2]VIP competitively and selectively inhibited VIP-stimulated adenylate cyclase activity (Ki = 0.1 microM). On male rat anterior pituitary homogenates the order of potency of the peptides was VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP. [D-Ser2]VIP and [D-His1]VIP acted as partial agonists. Besides, [D-Phe2]VIP and [D-Arg2]VIP were inactive as well as unable to inhibit VIP-stimulated adenylate cyclase activity. These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N-terminal moiety of the molecule.

Anterior pituitary glandular kallikrein: Trypsin activation and estrogen regulation

Discrepant reports exist regarding the presence of glandular kallikrein or other trypsin-like serine proteases in the pituitary. The existence of pituitary kallikreins in latent forms could explain these discrepancies. I report that trypsin treatment of rat anterior pituitary homogenates activates two serine proteases which generate kinins from kininogen and selectively cleave chromogenic substrates for kallikreins. One protease (enzymatically and immunologically resembling glandular kallikrein) and activated 5-fold by trypsin and was 20 times more abundant in female than in male lobes due to hormonal regulation by ovarian estrogens. The second kallikrein (activated 20-fold by trypsin) was unaffected by estrogens. The results demonstrate that rat anterior pituitary kallikreins predominantly exist in latent forms requiring activation for detection. Additionally, glandular kallikrein is a major estrogen-induced protein in the rat anterior pituitary. No other member of this large protease family is known to be regulated by estrogens.

Pituitary binding of 3H-labeled Sertoli cell factor in vitro: a potential radioreceptor assay for inhibin.

We have previously demonstrated that binding of partially purified, 3H-labeled Sertoli cell factor (SCF) to rat anterior pituitary homogenates was tissue specific, saturable, time and temperature dependent, and competitively inhibited by unlabeled SCF. The present study further characterized the binding of [3H]SCF to rat anterior pituitary in vitro and explored its potential use as a radioreceptor assay for SCF and other inhibin preparations. [3H]SCF was synthesized by rat Sertoli cells cultured in the presence of [3H]leucine (5 mu Ci/ml) and was then partially purified by Sephadex gel filtration and high pressure liquid chromatography. The purified [3H]SCF had a specific activity of approximately 20,000 dpm/micrograms protein and was biologically active in pituitary cell cultures. The binding was carried out in 0.5 ml buffer, containing one pituitary equivalent and, wherever appropriate, various unlabeled competing substances. The binding was optimal at pH 7.4 and was decreased by pretreatment of [3H]SCF with trypsin (0.25%; 37 C; 30 min) or heat (100 C; 10 min). Storage of the pituitary glands at -20 C for several months and differences in animal age did not affect total binding per pituitary, but the amount of radioactivity bound per mg pituitary tissue declined progressively between 18-90 days of age. Over 90% of the bound [3H]SCF was competitively inhibited by excess unlabeled SCF and several other inhibin preparations of testicular origin: high mol wt fraction of ram testis fluid (mol wt, greater than 10,000), low mol wt fraction of ram testis fluid (mol wt, less than 5,000), ovine testicular lymph, and aqueous rat testicular extract. The degree of inhibition was dose dependent, and except for the low mol wt fraction of ram testis fluid, the displacement curves were parallel (slope, 0.95). In contrast, various noninhibin substances tested [rat androgen-binding protein, bovine LH, BSA, native GnRH, or GnRH agonist analogs D-Ser-(tBu)6-des-Gly10-GnRH-N-EA and D-Ala6-des-Gly10-GnRH-N-EA] did not significantly compete for the [3H]SCF binding. The binding ability correlated well with inhibin biological activity in vitro. These results provide additional evidence for the presence of SCF-binding sites in the rat anterior pituitary which interact with several different inhibin preparations of testicular origin but appear to be distinct from GnRH-binding sites. Our results also indicate that the pituitary binding may be used as a rapid radioreceptor assay for SCF and various other inhibin preparations.

Characterization of a peptide alpha-amidation activity from rat anterior pituitary.

A secretory granule associated enzymatic activity from the rat anterior pituitary that can convert the synthetic peptide D-Tyr-Val-Gly into D-Tyr-Val-NH2 is described. Due to the presence of inhibitory activity in the cytosol and rough endoplasmic reticulum/Golgi apparatus fractions, the alpha-amidation activity is barely detectable in crude rat anterior pituitary homogenates. The alpha-amidation activity was primarily soluble, displayed a pH optimum of about 7.0, and showed a requirement for molecular oxygen. The activity was stimulated 7-fold by the addition of optimal concentrations of copper. Of the cofactors tested, only reduced ascorbate produced a severalfold stimulation of activity. Studies with varied D-Tyr-Val-Gly or varied monoiodo-D-Tyr-Val-Gly demonstrate Michaelis Menten kinetics with indistinguishable kinetic constants for both peptides. Upon addition of ascorbate, the apparent Km and Vmax for the synthetic substrate, as estimated from Lineweaver-Burk and Eadie-Hofstee plots, increased by 30- and 60-fold, respectively. Several alpha-melanotropin- and gamma-melanotropin-related peptides with COOH-terminal glycine residues were effective competitive inhibitors of the reaction while the corresponding alpha-amidated peptides were very poor inhibitors. The rat anterior pituitary alpha-amidation activity appears to be very similar to the alpha-amidation activity in rat intermediate and neural pituitary, and mouse anterior pituitary tumor cells.

Subcellular distribution of particle-bound neutral peptidases capable of hydrolyzing gonadoliberin, thyroliberin, enkephalin and substance P.

Subcellular fractions from rat anterior pituitary homogenates were obtained by differential and gradient centrifugation, identified with the help of marker enzymes and screened for peptidases capable of hydrolyzing gonadoliberin, thyroliberin, enkephalin and substance P. Since each neuropeptide is susceptible to cleavage by more than one enzyme, specific substrates or inhibitors have been used for the selective determination of the individual peptidasic activities. Among the various enzymes tested, the angiotensin-converting enzyme, the thermolysin-like metalloendopeptidase ('enkephalinase'), a thyroliberin-degrading enzyme and some aminopeptidasic activities were found to be associated with the plasma membrane. Other aminopeptidases, a gonadoliberin-degrading and a substance-P-degrading enzyme are associated with the mitochondria and thus are most likely not involved in the biological inactivation of neuropeptides.

Reciprocal relation between serum thyrotropin levels and intrapituitary 3,5,3'L-triiodothyronine generating activity from thyroxine in perinatal rats.

Thyroxine (T4) 5'-monodeiodinating activity in rat pituitary was studied, using paper chromatography. Rat anterior pituitary homogenates were incubated with 125I-T4, dithiothreitol and unlabeled L-T4 at 37 degrees C. The incubation mixture was extracted by ethanol and the extracts were subjected to descending paper chromatography. The conversion ratio of T4 to 3, 5, 3'L-triodothyronine (T3) was calculated from the radioactivity of T3 spot to total radioactivity on the paper strip. The T3 amount generated was estimated from the T4 concentration in the incubation mixture and the conversion ratio. The T3 generating activity from T4 increased as the amount of tissue increased. It was temperature-and pH-dependent, and thiol sensitive. These results suggest the enzymatic nature of T4 5'-monodeiodinating activity of the anterior pituitary. A kinetic study revealed low Km for T4 (7.9 +/- 1.6 nM, Mean +/- SE), with Vmax of 68.0 +/- 12.7 fmoles T3/mg protein/min. T4 monodeiodinating activity was consistently, though minimally, detected in fetal rat pituitaries and increased after birth, reaching the maximum at 22 days. It declined thereafter to the young adult level. Serum TSH levels were markedly elevated in fetus. They decreased after birth, reaching the nadir at 22 days, and then increased to young adult levels. Serum T4 and T3 levels were markedly diminished in fetus and gradually increased after birth, reaching the young adult levels at 17 days. Thus, a reciprocal relationship was observed between intrapituitary T3 generating activity from T4 and serum TSH levels in developing rats. It suggests that the conversion of T4 to T3 in the pituitary plays a role in regulating TSH secretion.

Thyroxine 5'-monodeiodination in rat anterior pituitary homogenates.

The conversion of T4 to T3 by 5′-monodeiodination was studied in anterior pituitary homogenates. No T4 5′- monodeiodination was seen unless dithiothreitol (DTT) or reduced glutathione was added. DTT (5 mM) caused measurable conversion, and maximum rates occurred between 50–100 mM DTT. Glutathione was a less potent stimulant. T4 5′-monodeiodination was pH dependent, with a pH maximum of 7.5, and temperature dependent between 4–37 C. Preheating the homogenate to 75 C for 30 min destroyed more than 95% of the activity. The reaction rate was proportional to the tissue content in the homogenate. At submaximal but not at saturating DTT concentrations, incubations under nitrogen increased the reaction rate to 161% of the rates in incubations under air. There was no significant effect of 5 mM EDTA, 5′ M α-tocopherol, 100 mM KC1, 100 –M Nal, 140 μM TRH, 300 μM propylthiouracil, or 300 HM methimazole. Catalase (2000 U/ml) had a variable inhibitory effect depending on the DTT concentration. The T4 5′-monodeiodinatio...

Prolactin releasing effect of sulpiride isomers in rats and man.

Sulpiride, an antipsychotic drug of the benzamide class, reportedly displaces stereospecifically [3H]-butyrophenones from putative dopamine (DA) binding sites in rat striatum. To evaluate if sulpiride displays the same stereospecificity in the inhibition of pituitary DA receptors, the effect of the two (-)- and (+)-sulpiride isomers was tested with regard to their ability to stimulate prolactin (PRL) secretion in rats and man and to displace [3H]-spiroperidol bound to rat anterior pituitary receptors. In male rats, (−)-sulpiride at doses of 0.1 and 1.0 mg/kg i.p., induced a maximum PRL-releasing effect, not different from that evoked by a dose of 10 mg/kg of the compound. (+)-Sulpiride was active only at the dose of 10 mg/kg i.p., and its PRL-releasing effect was superimposable to that evoked by the same dose of (−)-sulpiride. Similarly, in 8 normal subjects (4 men and 4 women) only (−)-sulpiride was active as PRL releaser when the low dose of 0.25 mg i.v. was used; when the higher dose of sulpiride was used (4.0 mg i.v.), it induced a rise in plasma PRL of the same entity for both isomers at early post-injection times (15–30 min) but greater with the (−)-isomer at the following time intervals (45–120 min). (−)-Sulpiride displaced [3H]-spiroperidol bound to rat anterior pituitary homogenates with a potency about 100 times as greater as that showed by (+)-sulpiride. In all, these data indicate that sulpiride isomers display at the level of pituitary DA receptors for PRL control the same stereospecificity exhibited on a population of striatal DA receptors.

Binding of gonadotropin-releasing hormone (LH-RH) to the pituitary plasma membranes and the problem of adenylate cyclase stimulation

Specific binding studies of tritium-labeled LH-RH to sheep anterior pituitary membranes at 37°C showed a maximum of binding capacity of 110 ± 20 mol/mg protein with an association rate constant of 2 ± 1 × 10 5 M −1 s −1 and a dissociation rate constant of 0.7 ± 0.4 × 10 −1 s −1. The Scatchard plot data showed a single type of binding site with K ass = 1.1 ± 0.4 × 10 8 M −1 in good agreement with the kinetic studies. Various doses of LH-RH in the presence or absence of Ca 2+, were unable to stimulate adenylate cyclase either in the rat anterior pituitary homogenates, or in the purified sheep anterior pituitary plasma membranes. To explain these results, it may be argued that the proportion of gonadotrophs in the pituitary gland is too small to show a significant increase in the LH-RH-induced adenylate cyclase activity. Another possibility is the disconnection of the hormonal receptor from the site of activation of adenylate cyclase during the preparation of plasma membranes. Finally, cAMP may not be involved in LH release by LH-RH.