Distribution of angiotensinogen immunoreactivity in rat anterior pituitary glands

Angiotensin II (AII) has been previously shown to be localized in the gonadotropes of the rat anterior pituitary gland. Renin and angiotensin-converting enzyme, two enzymes that participate in the generation of AII, also have been shown to be present in gonadotropes. To determine whether angiotensinogen, the precursor to AII, is present in the same cells, we have stained rat anterior pituitary sections with an antirat angiotensinogen antiserum. Angiotensinogen staining was observed in cells that had a distinctive distribution at the periphery of the gland; the number of these cells and the intensity of the staining were increased in the pituitaries of rats that had been nephrectomized 24 hr before sacrifice. When double staining was performed, we never observed colocalization of angiotensinogen with any of the known pituitary hormones or with S100 protein. The results show that in the rat anterior pituitary gland, angiotensinogen is present, at least for the most part, in cells that are different from those containing renin, angiotensin-converting enzyme, and AII.     

Presence of ACTH-potentiating factors in rat anterior pituitary glands

High molecular weight ACTH fractions, obtained through gel filtration of boiled rat anterior pituitary extract, induced a marked increase in corticosterone production from isolated rat adrenal cells in the presence of low concentrations of ACTH-(1-24). This indicates the presence of heat-stable factors augmenting the steroidogenic action of ACTH in the rat anterior pituitary. We also noted that these factors potentiated the activity not only of ACTH-1(1-24) but also of ACTH-(1-8). The ACTH-potentiating factors in rat anterior pituitary extract are possibly present in heterogeneous forms according to their molecular weights (8,000, 10,000 and 15,000), their mobility in ion-exchange chromatography and their content in RIA-ACTH activity. Of these three forms, the former comigrated with biological ACTH activity. The remaining two forms were free of it. Since the effect of potentiating factors on modified ACTH-(1-9), shown to be less susceptible to proteolytic degradation from ACTH-(1-24), was similar to the effect on ACTH-(1-24), it is suggested that potentiation was not due to an inhibition of ACTH proteolysis.     

Vasopressin rapidly stimulates phosphatidic acid-phosphatidylinositol turnover in rat anterior pituitary cells

In cultured rat anterior pituitary cells, the agonist [Asu1,6, Arg8]vasopressin (AVP-A) increased by 1.5-fold 32Pi incorporation into phosphatidic acid (PA), as early as 15 s after its addition. Increased phosphatidylinositol (PI) labeling became significant 4 min after AVP-A addition. Dose-response measurements with AVP-A showed ED50 values of 76 and 62 nM for PA and PI labeling, respectively. Peptide corticotropin-releasing factor (CRF) (0.1 microM) did not affect the stimulatory effect of AVP-A on PA and PI labeling. These data suggest that stimulation of PI metabolism in corticotrophs may be one of the early events involved in the stimulation of ACTH release induced by vasopressin.     

Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

The ability of bombesin (BN)-like peptides to stimulate phosphatidylinositol turnover in rat brain slices was investigated. BN (1 μM) significantly stimulated inositol-1-phosphate (IP₁) but not inositol-4,5-biphosphate (IP₂) or inositol-1,4,5-trisphosphate (IP₃) production using frontal cortex slices in the presence of LiCl (7.5 mM); BN had no effect on cAMP or cGMP levels. BN and the structurally-related gastrin releasing peptide (GRP) elevated IP₁ levels in a dose-dependent manner. Similarly, nanomolar concentrations of the GRP fragment (Ac-GRP^20–27) significantly elevated IP₁ levels, whereas micromolar concentrations of the inactive GRP^1–16 did not. BN significantly elevated IP₁ levels in those brain regions enriched in BN receptors such as the olfactory bulb, hippocampus, striatum, thalamus and frontal cortex, whereas IP₁ levels were not significantly increased in areas which have a low density of BN receptors such as the cerebellum, medulla/pons and midbrain. These data suggest that CNS BN receptors may utilize phosphatidylinositol as a second messenger.     

Immunocytochemical localization of secretogranin III in the anterior lobe of male rat pituitary glands.

Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane.     

Effects of streptozotocin-induced hyperglycemia on agonist-stimulated phosphatidylinositol turnover in rat aorta

Altered vascular sensitivity and responsiveness has been previously described in various stages of experimental diabetes mellitus. Increases in membrane bound Ca2+ and intracellular calcium in diabetic aorta have been postulated to explain excitation-coupling dysfunction in diabetic vascular smooth muscle (VSM). Receptor-mediated phosphatidylinositol (PI) hydrolysis is known to activate VSM contraction; thus contractile changes in diabetic aorta could be functionally linked to abnormal PI turnover. To evaluate this possibility, parallel experiments were undertaken to study contractility and receptor-stimulated PI turnover with norepinephrine (NE), phenylephrine (PE), and serotonin (5-HT). At 7 and 28 days following injection of streptozotocin with production of the hyperglycemic state, aortas were harvested for contractile and PI turnover experiments. No differences in the contractile cumulative dose responses or receptor-mediated PI turnover were measured in the 7 day group. At 28 days, vascular supersensitivity and increased responsiveness were observed. PI hydrolysis in basal and agonist-stimulated aorta was, however, markedly decreased at 28 days. These findings suggest that mobilization and utilization of Ca2+ during contraction occur independently of receptor-stimulated PI hydrolysis in aorta from hyperglycemic rats. The duration of hyperglycemia also significantly effects contractility and PI turnover in rat aorta.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

Characteristics of the norepinephrine-stimulated phosphatidylinositol turnover in rat pineal cell dispersions

Dispersed rat pineal cells can be used for the study of the phosphatidylinositol effect. The response to (–)-norepinephrine of the incorporation of³²P_i into phospholipids is linear with time and cell concentration, stereospecific, and mediated through ₁-adrenergic receptors. Na⁺ in the incubation medium is obligatory for labeling of phosphatidylinositol and phosphatidylcholine by³²P. In the absence of K⁺ incorporation of³²P is drastically lowered and no stimulation by norepinephrine occurs. Rb⁺ can replace K⁺. Omission of Ca²⁺ or substitution with Sr²⁺ preferentially lowers incorporation of radioactivity into phosphatidylcholine. Mg²⁺ is not required for basal or stimulated labeling.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Piperine inhibits type II phosphatidylinositol 4-kinases: a key component in phosphoinositides turnover

Piperine has been shown to have anti-inflammatory properties. The molecular mechanisms by which it mediates anti-inflammatory activities remain elusive. Type II phosphatidylinositol 4-kinase(s) are key components in FcεRI receptor-mediated signaling leading to inflammatory mediators release in RBL-2H3 cells. The effects of piperine on IgE-mediated signaling and mast cell degranulation were investigated. Pretreatment of RBL-2H3 cells with piperine inhibited IgE-induced activation of type II PtdIns 4-kinase(s). In vitro lipid kinase assays showed piperine-inhibited type II PtdIns 4-kinase activity in a dose-dependent fashion with no effect on PtdIns 3-kinase activity. Concomitantly, pretreatment of RBL-2H3 cells with piperine also inhibited IgE-induced β-hexosaminidase release in RBL-2H3 cells. These results suggest that type II PtdIns 4-kinases are part of piperine-mediated anti-inflammatory signaling mechanisms.     

Functional ATP receptors in rat anterior pituitary cells

The effects ofATP and other nucleotides on the cytosolicCa²⁺ concentration([Ca²⁺]_i)of single immunocytochemically typed anterior pituitary (AP) cells havebeen studied. ATP increased[Ca²⁺]_iin a large percentage (60-88%) of all five AP cell types:lactotropes, somatotropes, corticotropes, gonadotropes, andthyrotropes. Additivity experiments suggest the presence of at leasttwo different receptors, one accepting both ATP and UTP (U receptor),producing Ca²⁺ release from theintracellular stores, and the other preferring ATP (A receptor),producing Ca²⁺ (andMn²⁺) entry. The characteristicsof the U and A receptors were consistent with those ofP2Y₂ andP2X₂, respectively, and theirdistribution in the different AP cell types was not homogeneous. Thepresence of other ATP receptors suchP2Y₁ orP2X₂/P2X₃heteropolymers in a small fraction of the cells cannot be excluded.Thus functional ionophoric P2X receptors, which are typical of neuraltissue, are also present in the pituitary gland and could contribute to regulation of the gland's function.