Anterior Pituitary Homogenates Research Articles

Pregnenolone Sulfate Modulates Angiotensin II-Induced Inositol 1,4,5-Trisphosphate Changes in the Anterior Pituitary of Female Rat

The present study was designed to test whether pregnenolone sulfate can influence the angiotensin II (AngII) action in the anterior pituitary. Female rats were ovariectomized and 10 days after operation were treated with either 50 or 250 μg per animal in one of the following substances: oil (control), pregnenolone sulfate (PREG-S), estradiol benzoate (E2,) progesterone (P), or dehydroepiandrosterone sulfate (DHEA-S), given in single intraperitoneal injection. Because AngII is known to act in the anterior pituitary through the phosphatidiloinositol breakdown, thus increasing the level of inositol-1,4,5-trisphosphate (IP3), the IP3 concentration was determined 24 h after the injection in the anterior pituitary homogenate after in vitro exposure to AngII. Among the tested steroids only PREG-S enhanced the basal (without AngII) and AngII-induced level of IP3 in both doses used. There was no difference between the effect of a high and low dose of PREG-S. These results show that PREG-S may modulate AngII action in the anterior pituitary, regardless of the presence of ovary.

Multiple tyrosine hydroxylase transcripts and immunoreactive forms in the rat: differential expression in the anterior pituitary and adrenal gland.

Several regulatory neurofactors, classically associated with the hypothalamus, may be synthesized in the anterior pituitary (AP). Dopamine (DA) is the main prolactin-inhibiting factor. Its de novo synthesis in the normal AP has not been proved, although the TH transcript has been previously demonstrated by RT/PCR in the AP. We investigated tyrosine hydroxylase (TH) gene expression at both the protein and mRNA levels in the AP of normal random cycling female rats and in a catecholaminergic tissue, the adrenal gland (AG). The Western blot analysis of AP homogenates revealed two immunoreactive forms of TH in the AP, both differing from the TH present in the AG. RT/PCR products from AP and AG mRNA were subcloned and sequenced. In addition to the full-length form, we identified two TH transcripts generated by alternative splicing either involving the use of a new alternate splice-donor site within exon 2 or skipping exon 11. The form lacking exon 11 was not isolated from the AG. In the AP, all three forms were present. Although the AP contained the full-length TH mRNA, the expected size protein was not detected. Thus, there is alternative splicing of the TH primary transcript, and putative additional post-translational regulation may yield TH proteins with no enzymatic activity, at least in non-catecholaminergic tissues.

Insulin-like growth factor-I (IGF-I) and IGF-binding proteins in bovine sera and pituitaries at different stages of the estrous cycle.

The objective of this study was to determine whether concentrations of hypophyseal and serum insulin-like growth factor-I (IGF-I) and binding activities of serum and hypophyseal IGF-binding proteins (IGFBPs) differ with stage of the estrous cycle in mature beef cows. Cows were assigned to the following stages of the estrous cycle based on serum concentrations of progesterone (P4) and ovarian structures at death: days 1-5 (day 0 = estrus; n = 18), days 6-10 (n = 24), days 11-16 (n = 39), and days 17-21 (n = 7). Serum samples collected at death and anterior pituitary homogenates were analyzed for IGF-I, LH, and FSH by RIA. Serum and pituitary IGFBPs were evaluated by ligand and immunoblot analyses. Serum samples contained IGFBP activity at 44 and 40 kilodaltons (kDa; IGFBP-3), 34 kDa (IGFBP-2), 30 kDa (IGFBP-5), 28 kDa, and 24 kDa. The intensity of binding by the different sized proteins in serum remained constant throughout the estrous cycle. IGFBPs detected in anterior pituitaries included a 36-/40-kDa doublet (IGFBP-3), a 32-kDa protein (IGFBP-2), and a 29-kDa IGFBP (IGFBP-5). The intensity of [125I]IGF-I binding to IGFBPs was greater (P < 0.05) during days 11-16 than days 1-5 or days 6-10 of the estrous cycle and was intermediate (P > 0.05) during days 17-21. Serum concentrations of LH were low (< 0.4 ng/ml) during days 1-16 of the estrous cycle, but increased (P < 0.05) approximately 3- to 4-fold during days 17-21. LH concentrations in the anterior pituitary increased (P < 0.05) from the postovulatory period (548 +/- 52 micrograms/g; days 1-5) to the late luteal phase (791 +/- 39 micrograms/g; days 11-16) and were intermediate (P > 0.05) during the preovulatory phase (707 +/- 85 micrograms/g; days 17-21). Concentrations of IGF-I and FSH in the anterior pituitary and serum did not differ (P > 0.05) by stage of the estrous cycle. A positive correlation among the different IGFBPs in the pituitary (P < 0.01) and between each pituitary IGFBP and serum P4 (P < 0.05) existed. The significance (P < 0.0002) of the correlation between pituitary IGFBP-3 and P4 was greater (P < 0.02) than that between the other pituitary IGFBPs and P4. In summary, IGFBP activity in the anterior pituitary, but not that in serum, changed with stage of the estrous cycle in association with serum concentrations of P4. The absence of similar changes in serum or anterior pituitary concentrations of IGF-I supports the hypothesis that IGFBPs may regulate the actions of IGF-I on gonadotropin release from the anterior pituitary gland during the estrous cycle of beef cattle.

Generation of 1-37 and 1-38 forms of adrenocorticotropin by mono- and dipeptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles.

ACTH is a 39-amino acid peptide synthesized in the pituitary as part of the precursor molecule, POMC. Analysis of bovine anterior pituitary homogenates and secretory vesicles revealed that in addition to ACTH-(1-39), ACTH-(1-37) and ACTH-(1-38) were also present in the lobe, indicating that carboxyl-terminal processing of ACTH-(1-39) occurred in vivo. Mono- and dipe;tidyl carboxypeptidase activities that cleaved ACTH-(1-39) were detected in bovine intermediate and anterior pituitary secretory vesicle membranes and characterized. The dipeptidyl carboxypeptidase activity liberated ACTH-(1-37) and the dipeptide, Glu-Phe, and the monocarboxypeptidase activity generated, to a smaller extent, ACTH-(1-38) and phenylalanine from ACTH-(1-39). Kinetic studies indicated that the formation of ACTH-(1-37) occurred within minutes, whereas the formation of ACTH-(1-38) occurred within hours. Both enzymatic activities had a pH optimum of 5.5 and a Km of 14-18 microM for ACTH-(1-39), and were inhibited by serine and some thiol, but not metallo- or aspartic protease inhibitors. These unique serine carboxypeptidase(s) may function as a converting enzyme in vivo.

Generation of 1-37 and 1-38 forms of adrenocorticotropin by mono- and dipeptidyl serine carboxypeptidase activities in bovine pituitary secretory vesicles

ACTH is a 39-amino acid peptide synthesized in the pituitary as part of the precursor molecule, POMC. Analysis of bovine anterior pituitary homogenates and secretory vesicles revealed that in addition to ACTH-(1-39), ACTH-(1-37) and ACTH-(1-38) were also present in the lobe, indicating that carboxyl-terminal processing of ACTH-(1-39) occurred in vivo. Mono- and dipe;tidyl carboxypeptidase activities that cleaved ACTH-(1-39) were detected in bovine intermediate and anterior pituitary secretory vesicle membranes and characterized. The dipeptidyl carboxypeptidase activity liberated ACTH-(1-37) and the dipeptide, Glu-Phe, and the monocarboxypeptidase activity generated, to a smaller extent, ACTH-(1-38) and phenylalanine from ACTH-(1-39). Kinetic studies indicated that the formation of ACTH-(1-37) occurred within minutes, whereas the formation of ACTH-(1-38) occurred within hours. Both enzymatic activities had a pH optimum of 5.5 and a Km of 14-18 microM for ACTH-(1-39), and were inhibited by serine and some thiol, but not metallo- or aspartic protease inhibitors. These unique serine carboxypeptidase(s) may function as a converting enzyme in vivo.

B-HT 920 activates dopamine D2 receptors coupled to inhibition of adenylate cyclase activity.

In homogenates of female rat anterior pituitary, the azepine derivative B-HT 920 inhibited the forskolin-stimulated adenylate cyclase activity with an EC50 value of 0.35 microM. In male rat anterior pituitary, B-HT 920 curtailed the stimulation of adenylate cyclase activity by vasoactive intestinal peptide with an EC50 of 0.20 microM. In synaptic plasma membranes of rat striatum, B-HT 920 significantly reduced basal adenylate cyclase activity with an EC50 of 0.68 microM. Both in pituitary and striatum, the B-HT 920 inhibition was counteracted by the dopamine (DA) D2 receptor antagonist 1-sulpiride, but not by the alpha 2-adrenergic antagonist yohimbine. These results indicate that B-HT 920 is capable of activating DA D2 receptors negatively coupled to adenylate cyclase activity.

Atipamezole, benzodiazepines, bicucullin and tifluadom antagonize the effect of TRH on rat duodenum and displace it from brain and anterior pituitary receptors.

The mechanism of action of the TRH induced inhibition of contractions of the transmurally stimulated rat duodenum has been studied. The effect of TRH was not antagonized by atropine, pentolinium, phenoxybenzamine, sotalol, methysergide, domperidone, diphenhydramine, cimetidine, aminophylline, antazolin, indomethacin, morphine, naloxone or tetrodotoxin. In contrast, the adrenergic alpha 2-antagonist atipamezole, the benzodiazepines chlordiaxepoxide and midazolam or GABA-A-antagonist bicucullin but not picrotoxin or SR-95531 attenuated the response to TRH. An opioid-kappa-receptor agonist having benzodiazepine structure, tifluadom, but not MR 2034 also diminished the response to TRH. However, these actions were not modified by the alpha 2-agonist medetomidine, benzodiazepine antagonist flumazenil, GABA-agonists muscimol or baclofen or naloxone, respectively. While the binding of [3H][3-Me-His2]TRH to the rat anterior pituitary, hypothalamus, cortex and brainstem homogenates was saturable and of high affinity, no saturable binding was observed in the duodenal smooth muscle. Agents that were effective in the duodenal preparation displaced [3H][3-Me-His2]TRH from its binding sites in brain homogenates and the inhibitory constants (Ki) were (in microM): 0.038-0.107 (TRH), 0.19-5.8 (chlordiazepoxide), 0.021-8.9 (midazolam), 1.5-17 (tifluadom), 60-210 (bicucullin) and 150-530 (atipamezole). Atipamezole, bicucullin and chlordiazepoxide caused competitive displacement indicated by the increased KD of the labelled ligand but no change in the Bmax while tifluadom increased KD and decreased Bmax. It is concluded that the inhibitory effect of TRH on the contractions of the duodenal smooth muscle is mediated directly by the smooth muscle and it is apparently specific for TRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Norvaline 2-TRH: binding to TRH receptors in rat brain homogenates

Norvaline 2-thyrotropin-releasing hormone ([Nva 2]TRH) has been described as a thyrotropin-releasing hormone (TRH) analog with no thyrotropin (TSH)-releasing capacity but enhanced analeptic activity compared with TRH, as shown by the reversal of haloperidol-induced catalepsy. We have evaluated the receptor-binding properties of [Nva 2]TRH in homogenates of rat anterior pituitary, hypothalamus, brainstem and cortex tissue, using [ 3H]TRH and [ 3H][3-Me-His 2]TRH as radioligands. Apparent K i values at high affinity TRH-binding sites, labelled predominantly by [ 3H][3-Me-His 2]TRH, ranged from 17.0 to 36.9 μM in all tested regions. Additionally, [Nva 2]TRH was shown to compete with [ 3H]TRH at low affinity TRH-binding sites with similar affinities. It is concluded that the loss of TSH-releasing activity of [Nva 2]TRH appears to be due to a drastic reduction in binding affinity to the high affinity TRH receptor subtype. Its analeptic activity, however, may be mediated by low affinity TRH binding sites which are predominantly labelled by [ 3H]TRH or by yet unidentified mechanisms.

Substance P and related tachykinins induce receptor-mediated hydrolysis of polyphosphoinositides in the rat anterior pituitary

In the present study characterization of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIP 2-PLC) activity and receptor-mediated hydrolysis of PIP 2 in rat anterior pituitary membranes were investigated. Incubation of the membrane fraction of anterior pituitary homogenate with [ 3H]inositol-labeled PIP 2 in the presence of calcium increased the concentration of the water-soluble degradation product inositol trisphosphate (IP 3) in a time-dependent manner. PIP 2-PLC in the rat anterior pituitary had a pH optimum at 5.5 and a requirement for cations. Ca 2+ and Mg 2+ could activate the enzyme. Activity was maximal at a total magnesium concentration of 1 mM and at a free Ca 2+ concentration of 100 μ.M. The addition of the detergent Triton X-100 (0.05% w/v) to the membrane fraction resulted in a 50% decrease of PIP 2-PLC activity, whereas the presence of sodium deoxycholate (1 mg/ml) in the membrane fraction increased the PIP 2-PLC activity by 100%. The tachykinins substance P, 8-Tyr-substance P, physalaemin, neurokinin A, eledoisin, kassinin and neurokinin B induced receptor-mediated breakdown of [ 3H]inositol-labeled PIP 2 in the membrane fraction in a concentration-dependent manner, but with different potencies. The tachykinins displayed the following rank order of potencies: substance P > 8-Tyr-substance P > physalaemin > neurokinin A > eledoisin > kassinin > neurokinin B, which is consistent with the involvement of a NK-1 receptor. Combined treatment of anterior pituitary membranes by substance P and thyrotropin-releasing hormone (TRH) resulted in an additional increase in PIP 2-PLC activity compared to stimulation with TRH alone.

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.

Second messengers associated with the action of AII and dopamine D2 receptors in anterior pituitary. Relationship with prolactin secretion.

We have shown that DA receptors of the D2 subtype inhibit prolactin release by several mechanisms. DA receptors inhibit cyclic AMP production through a GTP binding protein sensitive to the Bordetella pertussis toxin. However, this mechanism cannot be involved in the blockade of the AII stimulated prolactin secretion by DA. This blockade is probably partly due to the inhibition of the AII-stimulated inositol phosphate production by DA. This inhibition is also sensitive to the Bordetella pertussis toxin. The toxin is able to ADP-ribosylate three substrates in anterior pituitary cells (39, 40 and 41 kDa). In addition, we show here that AII receptors inhibit adenylate cyclase of anterior pituitary cell homogenates, but not in intact cells.

Subcellular distribution of dynorphin-like immunoreactivity in rat adenohypophysis in comparison with luteinizing hormone and follicle-stimulating hormone

Dynorphin and other proenkephalin B-derived peptides exist in the rat adenohypophysis in high concentrations and may have important roles in endocrine function. At the cellular level, dynorphin peptides are colocalized with the gonadotropins in at least a subpopulation of gonadotrophs. In this study dynorphin-containing particles were compared with secretory granules containing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by means of differential centrifugation and sucrose density gradient centrifugation. When anterior pituitary homogenate of male rats was subjected to differential centrifugation, about 70% of both dynorphin- and LH-containing particles sedimented at 30,000 × g. LH granules and dynorphin-containing particles comigrated in continuous sucrose density gradients both under nonequilibrium conditions as well as when equilibrium was attained. FSH storage granules were found to sediment in slightly denser fractions, with substantial overlap. Hence, dynorphin-containing particles and gonadotropin-containing granules exhibit similar characteristics. These hormones may, therefore, be colocalized also at the subcellular level or stored in separate but similar vesicles.

Immunoaffinity chromatography of bovine FSH using monoclonal antibodies.

Bovine FSH (bFSH) was used to immunize BALB/c mice. Spleen cells were fused to the SP 2/0 cell line to produce hybridomas that secreted monoclonal antibodies to bFSH. One of these antibodies (USDA-bFSH-MC28) was extensively characterized and found to be a gamma 1 with kappa light chains, having extremely low cross-reactivity with other bovine pituitary hormones and with ovine and porcine FSH. The dissociation constant as measured by Scatchard analysis was 4.3 nmol/l, and proved to be in a very useful range for affinity chromatography. In an essentially one-step immunoaffinity chromatography procedure, bFSH was easily isolated in a single chromatographic step from crude anterior pituitary homogenate with better yield and with the same purity as classical chromatographic techniques.

Glandular kallikrein levels in the rat anterior pituitary during the estrous cycle and pregnancy.

Glandular kallikrein (a trypsin-like serine protease) is a major estrogen-induced protein in the rat anterior pituitary, which appears to be associated with lactotropes. The present study examined glandular kallikrein levels in the anterior pituitary during the rat estrous cycle and pregnancy. After trypsin treatment of anterior pituitary homogenates (to activate latent forms of the enzyme), glandular kallikrein activity was measured by using the chromogenic substrate D-val-leu-arg-p-nitroanilide: 98-95% of the enzymatic activity was immunoprecipitable with glandular kallikrein antiserum. Glandular kallikrein levels did not change significantly during the various phases of the rat estrous cycle. However, a sharp decrease was observed starting on Day 15 of pregnancy and lasting through parturition; levels had almost returned to control values by Day 5 of lactation.

Effect of calcium on adenylate cyclase of rat anterior pituitary gland.

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal C 19-5-ene steroids induce full estrogenic responses in rat pituitary gonadotrophs

Previous studies have shown that the C 19 adrenal steroid 5-androstene-3β,17β-diol (5-ene-diol), a metabolite of dehydroepiandrosterone (DHEA), can stimulate typical estrogenic responses in target tissues. Since estrogens are known to cause a specific stimulatory effect on LHRH-induced LH release in rat anterior pituitary cells in culture, we have taken advantage of the precision of this system to study the effect of 5-ene-diol or DHEA on this precise estrogen-sensitive parameter. Pretreatment for 48 h with 17β-estradiol (E 2), 5-ene-diol or DHEA induces a 2.4-, 2.7- and 2.6-fold stimulation of LH release induced by 0.3 nM LHRH, the effect being exerted at respective 50% maximally effective concentrations (ED 50 values) of 0.015, 45 and 115 nM. Following a 48-h preincubation with 10 nM E 2, 1 μM 5-ene-diol or 1 μM DHEA, the maximal LH and FSH responses to LHRH are increased by approx 50% above control. On the other hand, the sensitivities of the LH and FSH responses to LHRH as assessed by ED 50 values of LHRH action are increased by 3.3- to 7.5-fold. As further proof of the estrogenic nature of the effect of 5-ene-diol and DHEA, the effects of E 2, 5-ene-diol and DHEA are inhibited competitively by simultaneous incubation with the antiestrogen LY 156758 (keoxifene). The 2-fold stimulation of LHRH-induced LH release caused by DHEA-S, at concentrations within the range found in the plasma of women, is also completely blocked by 120 nM LY 156758. in direct binding studies, 5-ene-diol and DHEA or DHEA-S have approx 85- and ⪢ 10,000 lower affinities than E 2, respectively, for the estrogen receptor in rat anterior pituitary homogenate and human breast carcinoma cytosol. The present data clearly show that 5-ene-diol, DHEA and DHEA-S can exert full estrogenic activity in rat gonadotrophs, thus supporting the potential estrogenic role of these C 19 adrenal steroids in estrogen-dependent processes, especially breast cancer.

Identification of Neurotensin Receptors Associated with Calcium Channels and Prolactin Release in Rat Pituitary

Neurotensin (NT) is now reasonably well established as a neurotransmitter or neuromodulator candidate in the CNS. In the present study, we characterized the NT receptors in dispersed cells from the anterior lobe of rat pituitary and investigated the involvement of both cyclic AMP and calcium in the release of prolactin (PRL) induced by NT receptor stimulation. The [3H]NT binding to membranes from anterior pituitary dispersed cells was found saturable and stereospecific. Scatchard analysis of the data gave a straight line indicating a Bmax value of 121 +/- 11 fmol/mg protein and a KD value of 1.4 +/- 0.2 nM. The calculated IC50 values for [3H]NT binding were 5.8 nM for NT, 7.8 nM for L-Phe-NT, and 3,000 nM for the pharmacologically inactive form D-Phe-NT. NT, up to a concentration of 1 microM, did not affect the cyclic AMP generating system in homogenates of anterior pituitary from male or lactating female rats. The same pattern of results was obtained for cyclic AMP formation in intact cells. NT and its analogs stereospecifically enhanced the influx of calcium into dispersed cells from rat anterior pituitary. The effect was time- and dose-dependent. It appeared to be associated with neurotransmitter-operated calcium channels since: preincubation of the cells with tetrodotoxin did not affect the increase in calcium influx induced by NT; concentrations of verapamil that counteract the influx of calcium induced by potassium lacked the capacity to modify the influx of calcium induced by NT; and NT lost its capacity to release PRL in the absence of extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Atrial natriuretic factor does not affect basal, forskolin- and CRF-stimulated adenylate cyclase activity, cAMP formation or ACTH secretion, but does stimulate cGMP synthesis in anterior pituitary

The report that ANF inhibits basal and CRF-stimulated adenylate cyclase activity in anterior pituitary homogenates suggested that the atrial peptide could inhibit ACTH secretion. This possibility was investigated in the ACTH-secreting AtT-20 mouse pituitary tumor cell line as well as homogenates or primary cell cultures from rat anterior hypophysis. ANF (up to 5 × 10 −7 M) was found to be completely ineffective in stimulating basal, CRF- and/or forskolin-stimulated adenylate cyclase activity, cAMP accumulation and ACTH secretion. Similarly, ANF had no effect on spontaneous or GRF-induced GH release from cells in primary culture. ANF receptors, however, are present in AtT-20 cells and anterior pituitary cells as evidenced by the ability of the peptide to stimulate intracellular cGMP accumulation. The data, therefore, suggests that ANF does not have a negative modulatory action on the secretory function of anterior pituitary. The role of cGMP in any other action(s) of ANF remains unknown.