Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of hepatitis A virus.

Two RNA fragments from the region just upstream of the internal ribosome entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the U*U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+base pairs at neutral pH, but can apparently form them at pH values below 6. We present a series of possible models in which chain folding draws the strand termini closer together, possibly serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.

A novel hypothalamic peptide, pituitary adenylate cyclase-activating peptide, regulates the function of rat granulosa cells in vitro.

Pituitary adenylate cyclase-activating peptide (PACAP) is a novel peptide that was isolated from ovine hypothalamic tissue on the basis of its ability to stimulate cAMP accumulation in cultured rat pituitary cells. Recently we demonstrated that PACAP can stimulate cAMP accumulation and secretory function in cultured rat Sertoli cells. Since ovarian granulosa cells share many properties with Sertoli cells, we have examined the effect of PACAP (consisting of 38 or 27 amino acid residues) on cultured granulosa cell function. Granulosa cells were obtained from the ovaries of 25-day-old rats implanted with a silastic capsule containing diethylstilbestrol 5 days prior to culture. PACAP 38 (0.1 microM-0.01 pM), both alone and in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine, stimulated cAMP accumulation 4-8-fold with an ED50 of approximately 100 pM. Maximal PACAP 38 or PACAP 27 stimulation of granulosa cell cAMP was significantly greater than that produced by a maximally effective concentration of FSH. Because PACAP 38 and 27 have 68% sequence homology with vasoactive intestinal peptide (VIP), and since VIP stimulates granulosa cell cAMP accumulation and estradiol and progesterone secretion, we examined the possibility that PACAP could be acting via the VIP receptor. VIP stimulated cAMP only at concentrations of 10 nM or greater, whereas the PACAP stimulation was evident at 10 pM. Moreover, only one of three potent VIP antagonists inhibited VIP stimulation of cAMP accumulation, and only at 1 microM or greater. This VIP antagonist did not inhibit PACAP 38 action at 2000-fold excess concentration. Interestingly PACAP 38 was more effective than PACAP 27 with regard to steroid secretion and the ability to induce LH responsiveness. PACAP and VIP stimulation of granulosa cell cAMP accumulation or estradiol or progesterone secretion was not additive. Thus, these data support the hypothesis that granulosa cells have specific PACAP 38 receptors and that VIP acts via these receptors. In addition, PACAPs 38 and 27 are more potent stimulators of cAMP accumulation in luteinized granulosa cells than LH. These results both pre- and postovulation, along with previous data indicating that the PACAPs are found in the ovaries, suggest a role for PACAP in the regulation of ovarian function.

Specific inhibition of FSH-stimulated cAMP accumulation by delta 9-tetrahydrocannabinol in cultured rat granulosa cells.

delta 9-Tetrahydrocannabinol (THC), the major psychoactive component in marihuana has been shown to affect male and female reproduction in both humans and animals. THC has effects at the level at the pituitary-hypothalamic axis as well as a direct effect on the testis and ovary. A specific gonadal effect of THC is inhibition of steroidogenesis in cultured granulosa cells from immature, estrogen-treated rats that depends in part on a post-cyclic AMP site of action. The effect of THC on cAMP accumulation itself, however, has not yet been examined in this cell type. The aim of the present study was to examine the effect of THC on granulosa cell cAMP accumulation, a process that is important for several granulosa cell functions, including steroidogenesis. THC inhibited FSH-stimulated cAMP accumulation in a dose-dependent manner at concentrations which were not cytotoxic. This inhibition was evident by 2 hr and did not affect the dose of FSH which gave half-maximal stimulation, suggesting that THC does not compete with FSH for binding to its receptor. Detailed investigations regarding the mechanism of THC inhibition were conducted using forskolin and isoproterenol. In contrast to its effect on FSH stimulation, THC did not inhibit forskolin- or isoproterenol-stimulated cAMP accumulation, indicating a specific effect for the FSH receptor. In addition, inhibition of FSH-stimulated cAMP accumulation occurred in the presence of a phosphodiesterase inhibitor, indicating that THC does not decrease cAMP accumulation through an increase in the cAMP degradative pathway. These data indicate that a part of the reproductive toxicity seen with THC may be due to a specific alteration of the hormonal control of granulosa cell function via a specific inhibition of FSH-stimulated cAMP accumulation.