Homologous down-regulation of gonadotropin-releasing hormone receptor sites and messenger ribonucleic acid transcripts in alpha T3-1 cells.

GnRH is known to down-regulate its pituitary receptors by mechanisms that include endocytosis of the agonist-receptor complex. To evaluate the extent to which changes in receptor synthesis contribute to this process, the effects of GnRH and its analogs on GnRH receptor number and messenger RNA (mRNA) levels were analyzed in the alpha T3-1 gonadotroph cell line. Treatment with GnRH or its potent agonist analog, des-Gly10-[D-Ala6]GnRH N-ethylamide, reduced GnRH receptor number in a time- and dose-dependent manner, with a half-maximal decrease in response to 10(-6) M GnRH or agonist analog by 75 min. The maximum decrease in receptor number (to 31% of the control value) was sustained for up to 72 h. In alpha T3-1 cells incubated with 10(-8) M GnRH or agonist analog, the GnRH receptors fell by 28% and 46% after 2 h, respectively; no change in receptors occurred after treatment with 10(-8) M GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]GnRH). Time- and dose-dependent reductions in the level of receptor mRNA were also observed after treatment of alpha T3-1 cells with GnRH and the agonist analog. However, the maximal reduction in mRNA levels (to 60-70% of the control value) was consistently less than the decline in receptor number. These results indicate that the mechanism of GnRH receptor down-regulation in alpha T3-1 gonadotrophs includes reduction of receptor synthesis secondary to decreases in receptor mRNA levels. The finding that reductions in mRNA levels were relatively less than the decreases in receptor number is consistent with the involvement of additional mechanisms, including endocytosis and degradation, in down-regulation of the GnRH receptor.

Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes.

Synthesis of radiolabeled DNA probes via polymerase chain reaction (PCR) is a convenient alternative to the more conventional methods of random primer-labeling and nick translation. PCR requires less template and allows the synthesis of radiolabeled probes from specific sequences contained within cloning vectors and genomic DNA. Under nucleotide imbalance conditions where the concentration of the radiolabeled nucleotide was 0.825 microM and the other dNTPs were each > 25 microM, amplification by Taq DNA polymerase was inhibited. Reducing the concentrations of the unlabeled dNTPs resulted in greater yields of amplification product with maximal yield obtained when the concentration of three unlabeled nucleotides was two- to eightfold higher than that of the limiting labeled nucleotide. When we utilized this amplification method for synthesis of an 800-bp glyceraldehyde-3-phosphate (GAPDH) dehydrogenase probe, 87% of the added [32P]dCTP was incorporated into amplification product. Application of this method for synthesis of high specific activity probes ( > 4 x 10(9) cpm/micrograms) up to 2.6 kb in length is demonstrated and utility of the 800-bp GAPDH probe for hybridization to Northern blots for detection of GAPDH mRNA is presented.

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane.

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca2+ or Mn2+ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by 45Ca2+ influx). Mn2+ entry (fura2 quenching) was about 1.6 fold higher in internal Ca2+ pool-depleted acini (Ca(2+)-depl acini) than in unstimulated cells. Mn2+ entry into Ca(2+)-depl acini was increased at external pH > 7.4 and decreased at pH < 7.4. Pretreatment of Ca(2+)-depl acini with the relatively hydrophobic carboxylic group reagent, N,N'-dicyclohexylcarbodiimide (DCCD, 50 microM for 30 min) resulted in the inhibition of Mn2+ entry into Ca(2+)-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn2+ entry. Similar to the effects in intact acini, Ca2+ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca2+ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca2+ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca2+ and Mn2+ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.

Expression of gonadotropin-releasing hormone receptors and autocrine regulation of neuropeptide release in immortalized hypothalamic neurons.

The hypothalamic control of gonadotropin secretion is mediated by episodic basal secretion and midcycle ovulatory surges of gonadotropin-releasing hormone (GnRH), which interacts with specific plasma membrane receptors in pituitary gonadotrophs. Similar GnRH receptors and their mRNA transcripts were found to be expressed in immortalized hypothalamic neurons, which release GnRH in a pulsatile manner in vitro. Activation of these neuronal GnRH receptors elicited dose-related intracellular Ca2+ concentration responses that were dependent on calcium mobilization and entry and were inhibited by GnRH antagonists. Exposure of perifused neurons to a GnRH agonist analog caused a transient elevation of GnRH release and subsequent suppression of the basal pulsatile secretion. This was followed by dose-dependent induction of less frequent but larger GnRH pulses and ultimately by single massive episodes of GnRH release. The ability of GnRH to exert autocrine actions on its secretory neurons, and to promote episodic release and synchronized discharge of the neuropeptide, could reflect the operation of the endogenous pulse generator and the genesis of the preovulatory GnRH surge in vivo.

Glucose-regulated protein-78 expression in the rat adrenal cortex.

We have postulated that steroidogenesis activator polypeptide (SAP) is a product of glucose-regulated protein-78 (grp78) proteolysis on the basis of a number of considerations, including a striking sequence similarity between the carboxyl-terminal region of grp78 and SAP. Since ACTH stimulates the rapid intracellular accumulation of SAP, experiments were conducted to determine whether ACTH might also regulate levels of grp78 mRNA and/or protein. Using a grp78 cDNA probe, Northern analysis of total RNA isolated from hypophysectomized or dexamethasone-suppressed rats revealed that neither treatment had a measurable influence on steady-state levels of grp78 mRNA over a 4-day period. Moreover, immunoblotting with an antiserum directed against a shared grp78/SAP sequence failed to detect a significant change in the grp78 content of adrenal homogenates from dexamethasone-suppressed rats as compared with untreated controls. On the other hand, grp78 in cultured rat adrenocortical cells fell to 50% of that in time-zero controls after 72 h in the absence of ACTH, whereas inclusion of ACTH in the medium blocked this decline. We conclude that while adrenocortical grp78 may be under some measure of trophic ACTH control, the rapid fluctuations reported for SAP are not likely to be driven by large changes in the size of the grp78 pool.

Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor.

The primary structure of the gonadotropin-releasing hormone (GnRH) receptor was determined by sequencing a functional receptor cDNA isolated by expression cloning from an immortalized murine gonadotroph (alpha T3) cell line. Positive clone pools from a cDNA library were detected by screening expressed RNA in aequorin-injected Xenopus laevis oocytes, in which receptor-mediated calcium responses were monitored as light emission during stimulation by GnRH. The isolated receptor cDNA encodes a 327-amino acid protein that has seven putative transmembrane regions and is unique among G protein-coupled receptors in that the predicted sequence lacks a carboxyl-terminal cytoplasmic domain. COS-7 cells transfected with the receptor cDNA expressed high affinity binding sites for GnRH and its agonist and antagonist analogs and exhibited calcium responses to GnRH stimulation. These, and the prominent calcium responses of Xenopus oocytes injected with receptor RNA, were inhibited by GnRH antagonists. Northern blot analysis revealed two mRNAs (1.6 and 3.5 kilobases) in alpha T3 cells and in the mouse pituitary gland, and both transcripts were shown to encode functional GnRH receptors when expressed in Xenopus oocytes. In contrast, a single 4.6-kilobase receptor mRNA was present in rat anterior pituitary gland, ovary, and Leydig cells. The absence of a carboxyl-terminal cytoplasmic domain indicates the importance of other regions of the GnRH receptor in agonist-induced signal transduction, and possibly in receptor desensitization and sequestration.

Membrane potential modulates divalent cation entry in rat parotid acini.

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.

Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.

Adrenocorticotropin receptors: functional expression from rat adrenal mRNA in Xenopus laevis oocytes.

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A)+ RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. These were detected at 48 h and reached a maximum 72 h after microinjection of 20-40 ng of adrenal mRNA. In response to 1 microM ACTH, total cAMP production increased within 2.5 min and reached half-maximal and maximal levels (5-fold greater than basal) at 10 and 75 min, respectively, and then remained elevated for up to 5 h. Extracellular cAMP levels were much lower but showed prominent linear increases from almost undetectable levels, with 70- and 150-fold increases evident at 1 and 2 h, respectively. The half-maximal concentration (ED50) for stimulation of cAMP formation was 5 x 10(-8) M ACTH-(1-24); the ED50 for ACTH-(1-17) was 5 x 10(-7) M, and no response was observed with ACTH-(1-10). Size fractionation of rat adrenal poly(A)+ RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1- to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNA in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.

Refill status of the agonist-sensitive Ca2+ pool regulates Mn2+ influx into parotid acini.

We have utilized fura-2 and a Ca2+ surrogate, Mn2+, to assess the mechanism of Ca2+ entry involved in the refill of the internal agonist-sensitive Ca2+ pool in parotid acini. Both the muscarinic agonist, carbachol, and the alpha-adrenergic agonist, epinephrine, stimulate Mn2+ entry into dispersed parotid acini, which is detected as an augmentation in fura-2 fluorescence quench rate. The rate of Mn2+ entry into parotid acini, depleted of internal agonist-sensitive Ca2+ pools by prolonged carbachol stimulation in a nominally Ca2(+)-free medium, is not significantly changed by the addition of the muscarinic antagonist, atropine, but is significantly attenuated when these internal pools are allowed to either partially or totally reload with Ca2+. Also, we provide evidence which suggests that under conditions which promote refill, Mn2+ appears to directly enter the cytosol from the extracellular medium and is not accumulated into an internal Ca2+ pool either directly from the medium or via a cytosolic route. Thus, we suggest that during refill, Ca2+ enters into the cytosol prior to its recruitment into the internal agonist-sensitive Ca2+ pool and in turn, the magnitude of this entry is modulated by the refill status of this pool.

Calcium entry in rat parotid acini: activation by carbachol and aluminum fluoride.

Entry of extracellular Ca2+ into the cytosol of exocrine cells plays an important role in the process of fluid transport, especially during periods of prolonged secretion. However, in parotid acinar cells, the process of Ca2+ entry and the identity of factors which regulate it remain obscure. In this report, we demonstrate that AlF-4, like carbachol, activates Ca2+ entry into dispersed rat parotid acini. In physiological Ca2(+)-containing (1.28 mM) medium, both agents elicit three phases of cytosolic Ca2+ change, an initial transient increase (intracellular Ca2+ dependent) followed sequentially by a decrease (intra- and extracellular Ca2+ dependent) and a small sustained increase (extracellular Ca2+ dependent). Cytosolic Ca2+ concentration ([Ca2+]i) during the last two phases is influenced by variations in extracellular [Ca2+]. Elevation of extracellular [Ca2+], at any time after the initial transient increase, results in a rise of cytosolic [Ca2+], thus demonstrating the existence of a Ca2+ entry pathway during the two later phases. These data suggest the likelihood that in parotid acini, G protein activation is involved in stimulating this Ca2+ entry pathway. Because in AlF-4-treated acini entry into the cytosol is detectable only after the initial intracellular Ca2+ release phase, we suggest that this Ca2+ entry process does not accompany initial intracellular Ca2+ mobilization. Furthermore, the sustained cytosolic [Ca2+] elevation which can be observed 15-30 min after initial stimulation of acini is likely determined by this Ca2+ entry process which, in physiological conditions, could support sustained fluid secretion.

The kinetics of steroidogenesis activator polypeptide in the rat adrenal cortex. Effects of adrenocorticotropin, cyclic adenosine 3':5'-monophosphate, cycloheximide, and circadian rhythm.

The behavior of steroidogenesis activator polypeptide (SAP), a recently described modulator of cholesterol side-chain cleavage activity (Pedersen, R. C., and Brownie, A. C. (1987) Science 236, 188-190), was investigated in rat adrenocortical cells using a specific radioimmunoassay. In response to a maximal dose of adrenocorticotropic hormone (ACTH) (1 nM) or of 8-Br-cAMP (1 mM), an increase in intracellular SAP begins rapidly (less than 1 min) and reaches half-maximal and maximal levels (16-fold greater than basal) at 3 and 15 min, respectively. A plateau at this maximal concentration of SAP is then maintained. The levels of intracellular SAP content and of corticosterone output exhibit a similar dose-dependent response to ACTH (EC50 = 25 and 30 pM, respectively). Treatment of ACTH-stimulated cells with cycloheximide reverses the rise in SAP (t1/2 congruent to 5-7 min). In vivo the SAP content of adrenals from quiescent rats is concordant with the circadian rhythm of the pituitary-adrenal axis; at the apex (1800 h), adrenal SAP is 13-fold higher than at the nadir (0800 h), paralleling 2- and 7-fold variations in cholesterol side-chain cleavage activity and serum corticosterone levels, respectively. At both time points, SAP levels rise in response to stress. Of the rat tissues examined, only the major steroid-forming organs (adrenal cortex and gonads) had significant levels of immunoreactive, cAMP-responsive SAP, while cAMP-unresponsive immunoreactivity was also detectable in the thymus, spleen, and brain. Considered together with the biological activity previously demonstrated for SAP in vitro, these data are consistent with its role as a cAMP-dependent, cycloheximide-sensitive modulator of steroid biosynthesis.

Steroidogenesis activator polypeptide (SAP) in the guinea pig adrenal cortex.

Steroidogenesis activator polypeptide (SAP), a cytosolic stimulator of cholesterol side-chain cleavage (cholesterol SCC) previously characterized in the rat, was isolated from guinea pig adrenal cortex. This factor exhibited behavior on reverse-phase high-performance liquid chromatography (HPLC) that was indistinguishable from authentic SAP and crossreacted fully in a SAP radioimmunoassay. In dexamethasone-suppressed guinea pigs neither the concentrations of immunoreactive adrenal SAP nor the levels of cholesterol SCC activity were significantly different between the outer zones (zonae glomerulosa and fasciculata) and the inner zone (zona reticularis). However, at 10 min after treatment of dexamethasone-suppressed animals with ACTH1-24, the outer zone content of SAP was increased 42-fold over unstimulated controls whereas inner zone SAP was elevated only 4-fold. At the same time, cholesterol SCC activity was increased 2-fold in the outer zones but unchanged in the inner zone. In addition to SAP itself, a crossreacting 82 kDa protein (p82)--similar to the putative SAP precursor identified in the rat--was detected on two-dimensional immunoblots of guinea pig whole adrenal homogenate. There were no significant differences in the protein concentrations of p82 or of cytochrome P-450scc between zones, either with or without ACTH treatment. We conclude that the widely reported contrast in corticosteroidogenic potential between the zona fasciculata and the zona reticularis of the guinea pig may reflect a differential capacity to generate SAP, and thus activate cholesterol SCC, in response to ACTH.

Steroidogenesis activator polypeptide may be a product of glucose regulated protein 78 (GRP78).

Cholesterol side-chain cleavage is sensitive to antibiotic inhibitors of protein synthesis, suggesting that a labile protein may play a regulatory role in this process. We have previously characterized such a factor--steroidogenesis activator polypeptide (SAP). Given the low molecular weight of SAP (Mr 3215), a SAP precursor has been sought. Using immunoblotting techniques with two polyclonal antisera directed against portions of the SAP sequence, a single protein of apparent Mr 82,000 (p82) can be detected in rat adrenocortical tissue. Our data suggest that adrenal p82 is most likely the widely-distributed minor heat shock protein, glucose regulated protein 78 (GRP78). The two proteins share biochemical attributes, including pI (5.2) and ATP affinity, and the reported amino acid sequences for SAP and for the carboxyl-terminal end of GRP78 are nearly identical. We propose that SAP is cleaved from GRP78--or a cognate protein--and that this proteolysis is regulated in a manner characteristic of steroidogenic tissues.

Expression in Escherichia coli of a fusion protein product containing a region of the adenovirus DNA polymerase.

The bulk of an open reading frame extending from map coordinates 23.3 to 14.2 in region E2b of the adenoviral genome has been cloned and expressed from a chimeric plasmid in Escherichia coli. The cloning strategy used created a fusion protein of 124,000 daltons, which contained greater than 98% adenovirus-encoded sequences. Antiserum raised against this protein reacted with the authentic 140,000-dalton adenovirus DNA polymerase. Another serum raised against a synthetic hexapeptide whose sequence corresponded to the predicted carboxyl terminus of adenovirus-encoded DNA polymerase also reacted with the fusion protein and authentic adenovirus DNA polymerase. These results demonstrate that the cloned region of DNA encodes the adenovirus DNA polymerase.