A. Gravanis

Hepatocyte swelling leads to rapid decrease of the G‐/total actin ratio and increases actin mRNA levels

Exposure of isolated rat hepatocytes to hypotonic (190 mosinol/1) incubation media lowered the cellular G‐actin level without affecting the total actin content: here the G‐/total actin ratio decreased by 15.5 ± 1.4% (n=7). Similar effects were observed following isotonic cell swelling by either addition of glutamine (10 mM) or insulin (100 nM), resulting in a decrease of the G‐/total actin ratios by 13.5 ± 2.1 % (n = 5) and 14.1 ± 1.1% (n = 11), respectively. The effects of hypotonic exposure, glutamine and insulin on the G‐/total actin ratio largely occurred within 1 min and persisted for at least 2 h in presence of the respective effectors. After a 120 min exposure to hypotonic media, glutamine or insulin the actin mRNA levels were increased 2.4‐, 2.0‐ and 3.6‐fold, respectively. Hypertonic exposure lowered the G‐/total actin ratio by only 4.9 ± 2.5% (n = 4) and increased actin mRNA levels only 1.2‐fold. There was a close relationship between glutamine‐ and hypotonicity‐induced cell swelling and the decrease of G‐/total actin ratios. The data suggest that cell swelling exerts rapid and marked effects on the state of actin polymerization and increases actin mRNA levels. Thus, cytoskeletal alterations in response to cell swelling may be involved in the regulation of hepatic metabolism by cell volume.

Opioids transiently prevent activation of apoptotic mechanisms following short periods of serum withdrawal.

Abstract: Opioids exert a proapoptotic effect on several normal and tumoral cells. The aim of the present article was to examine the effect of opioids on the PC12 rat pheochromocytoma cell line, a model for the study of chromaffin cell apoptosis. These cells produce δ‐ and κ‐opioid agonists and their receptors. Our results were as follows: The κ‐ and δ2‐opioid receptor agonists had a rapid but transient effect on apoptosis at 3 h, whereas μ opioids did not. The effect of opioids was reversible by the opioid antagonists naloxone and nor‐binaltorphimine. The effect of opioids was protective, suppressing serum deprivation‐induced apoptosis to ∼50% of controls. The protective effect of opioids on PC12 apoptosis was measurable only under serum deprivation. The effect of opioids was remarkably reproducible and highly constant in timing, which did not appear to depend on the duration of the preceding serum deprivation. Finally, opioids prevented the elevation of the Bcl‐2 and Bak proteins following serum deprivation to the levels attained by serum supplementation. Our combined data suggest that opioids protect PC12 cells from entering a state of induced apoptosis following serum deprivation.

Cycle and age-related changes in corticotropin-releasing hormone levels in human endometrium and ovaries

Corticotropin-releasing hormone (CRH) is synthesized in most female reproductive tissues such as the ovaries and the uterus. In the non-pregnant uterus ,it is mainly produced by epithelial cells of the endometrium. Recent in vitro experimental findings show that endometrial CRH is under the positive control of progesterone ,participating in the decidualization process of endometrial stroma and the progression of blastocyst implantation. CRH is also produced in the thecal compartment of the human ovary ,controlling ovarian steroid hormone biosynthesis. In the present study we compared the concentration of immunoreactive CRH (ir-CRH) in biopsies from proliferative and secretory human endometria ,and from pre- and postmenopausal human ovaries. We found that the concentration of ir-CRH was significantly higher in the secretory (92 ± 8 pg/mg protein; n = 10) than the proliferative (75 ± 9 pg/mg protein; n = 12; p < 0.05) endometria. This observation supports the experimental in vitro findings associating endometrial CRH in intrauterine phenomena of the secretory phase of the menstrual cycle (decidualization and implantation). Additionally ,we have shown that the concentration of ir-CRH was significantly higher in the premenopausal (125 ± 12 pg/mg protein; n = 14) than the postmenopausal (100 ± 12 pg/mg protein; n = 12; p < 0.05) ovaries, suggesting that ovarian CRH is related to normal ovarian function during the reproductive lifespan.

The actin cytoskeleton in rapid steroid hormone actions

Early actin cytoskeleton reorganization is an important regulatory step of membrane‐initiated, non‐genomic steroid hormone actions. Specific intracellular signaling cascades control the rapid alterations of actin polymerization in a variety of cell models. Moreover, actin remodeling is a decisive component in the signaling of hormone‐induced early and late cellular responses. This article briefly summarizes the current knowledge on the steroid hormone‐induced early actin cytoskeleton rearrangements. It focuses on the current progress to characterize the glucocorticoid‐ and androgen‐initiated, tissue‐specific actin signaling pathways and discusses the plethora of cellular responses that are regulated by the early actin redistribution. It also provides insights into the potential clinical significance of actin dynamics and actin‐specific molecular signaling for nongenomic steroid hormone actions on tumor cells.

Expression and regulation of corticotropin‐releasing hormone binding protein (CRH‐BP) in rat adrenals

Corticotropin‐releasing hormone (CRH) is present in the adrenal gland acting as a paracrine factor via stimulation of the locally expressed CRH receptors. In this study, we examined if the adrenal CRH system also contains a key component of the neuronal CRH‐containing system, the CRH‐binding protein (CRH‐BP). Our data show that: (i) the CRH‐BP transcript is detectable using RT‐PCR in total RNA isolated from rat adrenals, and (ii) its protein product is also found by western blot analysis in cell lysates. (iii) Immunohistochemical staining showed that adrenomedullary chromaffin cells produce the bulk of adrenal CRH‐BP, an ability retained by the PC12 rat pheochromocytoma cell line. (iv) Regulation of adrenal CRH‐BP expression by major modulators of the CRH system was also examined. Protein expression appears to be under the positive control of CRH itself, protein kinase A effector cAMP, glucocorticoids and interleukin (IL)‐6. It is thus evident that CRH‐BP may play a role in mediating their effects in the adrenal. (v) Differentiation of PC12 into neuron‐like cells resulted in a significant increase in CRH‐BP, parallel to the induction of the CRH peptide itself. In conclusion, CRH‐BP mRNA and protein are present in normal rat adrenomedullary chromaffin cells and in the PC12 rat pheochromocytoma cell line, making the adrenal CRH system directly comparable with those described in the CNS.

Expression and characterization of Cys374 mutated human β-actin in two different mammalian cell lines: impaired microfilament organization and stability

Previous studies have demonstrated that addition of glutathione at the penultimate Cys374 residue of actin results in filaments with diminished mechanical stability. In the present work substitutions introducing a negatively charged (Asp and Glu) or a neutral (Ala) amino acid at position 374 of the human β‐actin and tagged at the N‐terminus with the flag epitope were studied by transient transfections into Ishikawa human endometrial and opossum kidney cells. Immunofluorescence revealed that microfilaments which incorporated negatively charged mutants were partially to severely disorganized when compared to the almost well‐formed actin‐Ala374 filaments or the wild type actin filaments. Furthermore, microfilaments containing either negatively charged mutant were more sensitive to the destabilizing action of cytochalasin B. In addition, Triton fractionation revealed a considerable reduction of flag‐actin content in the Triton insoluble fraction for cells expressing Asp374 or Glu374 mutant compared to wild type actin. These results demonstrate that negatively charged amino acid residues at the exposed C‐terminal tail strongly affect actin microfilament organization and dynamics in vivo.

Endometrial Corticotropin-Releasing Hormone: Expression, Regulation, and Potential Physiological Implications

Our findings show that human and rat uterus express the CRH gene. Epithelial cells of both species are the main source of endometrial CRH, while stroma does not seem to express it, unless it differentiates to decidua. Immunoreactive CRH, produced by endometrial cells, has the chromatographic characteristics of authentic hypothalamic CRH, while the size of its mRNA in both human and rat uterus is similar to or identical with its counterpart, present in placenta and hypothalamus (1.3 kb). Estrogens and glucocorticoids inhibit and prostaglandin E2 stimulates the promoter of human CRH gene in transfected human endometrial cells, suggesting that endometrial CRH gene expression is under the control of these agents. Moreover, in rats, endometrial CRH expression is significantly higher at implantation sites, compared to that at interimplantation uterine regions. Given the proinflammatory/vasoregulatory properties of CRH, we hypothesize that endometrial CRH may participate in the regulation of intrauterine phenomena, such as blastocyst implantation, endometrial vascularization, and myometrial contractility.

PC12 Cells as a Model to Study the Effects of Opioids on Normal and Tumoral Adrenal Chromaffin Cells

Normal adrenal chromaffin cells produce delta opioid peptides while at the same time they have mainly kappa opioid receptors. This paper describes our date regarding the expression of the prodynorphin gene, the precursor of a family of endogenous kappa opioid ligands, in the PC12 rat pheochromocytoma cell line, and the effects of synthetic kappa opioid agonists and of Naloxone on various aspects of PC12 cell function including their secretion of catecholamines, proliferation and differentiation. This is the first part of a series of projects aimed at studying, (a) the conditions under which the prodynorphin gene is expressed in normal adrenomedullary cells, and (b) the physiological role of the endogenous kappa opioids in the physiology of the adrenal medulla.

Anti‐STD Vaginal Contraceptive Sponges

Vaginal sponges offer women control over protection against both pregnancy and sexually transmitted diseases (STDs), including HIV. Spermicide-impregnated sponges combine the actions of a physical barrier that blocks the cervix with a material that absorbs the ejaculate and a spermicide. Commercially available spermicides contain 1-5% of nonoxynol-9, shown to inhibit organisms responsible for gonorrhea, chlamydia, candidiasis, genital herpes, syphilis, trichomoniasis, and HIV. On the other hand, nonoxynol-9 is associated with a significantly higher risk of vaginal colonization with bacterial agents, ulcerative genital diseases, and vulvitis. A lower dose of nonoxynol-9 appears to avert vaginal irritation without compromising contraceptive efficacy. Use of chlorhexidene, a spermicide less irritating to mucosal cells than nonoxynol-9 but active against HIV in vivo and in vitro, is under investigation. Also promising are initial findings regarding the Protectaid contraceptive sponge with F-5 gel. Epidemiologic studies and clinical trials should provide quantitative estimates of the level of protection offered by barrier methods and identify the method that combines the highest protection, ease of use, and user acceptability.