The follicle is the compartment of the ovary responsible for estrogen production. In the pig follicular estrogen production is accomplished by a concerted cooperation between the two cell types of the follicle, the granulosa cells, and the thecal cells, under the control of the pituitary gonadotropins Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH). Under the influence of LH stimulation, the thecal cells increase their production of the androgen, androstenedione. FSH stimulation of the enzyme aromatase, increases granulosa cell estradiol production from androstenedione provided by the thecal cells. Granulosa cells from large preovulatory follicles luteinize in vitro when placed in culture. This process is characterized by a decline in estrogen production and by increased progesterone production. To better define the steroid metabolic activity of granulosa cells during this period of in vitro luteinization, studies were performed to assess the metabolism of (A-dione) by granulosa cells in culture. The results of these studies demonstrated that aromatase activity of the granulosa cells declined substantially between 12 and 48 hours of culture resulting in the loss of estrogen production. In addition to this finding, an alternative pathway of A-dione metabolism was discovered. This pathway involves the production of the novel acidic steroid 19-oic-androstenedione (19-oic-A), and two additional novel metabolites, the C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids 5(10)estrene-3{dollar}\beta,\ 17\beta{dollar}-diol (estrenediol) and 19-nor-testosterone (19-nor-T). Evaluation of the time course of A-dione metabolism suggested that aromatase was responsible for the production of 19-oicA from A-dione. The data also suggested that changes in metabolism associated with luteinization of the granulosa cells resulted in the metabolism of 19-oic-A to estrenediol and 19-nor-T. Further investigation confirmed that aromatase was the enzyme responsible for the production of 19-oic-A from A-dione. The metabolism of 19-oic-A by granulosa cells was assessed, and the results demonstrated that this steroid was indeed the metabolic precursor of the two C{dollar}\sb{lcub}18{rcub}{dollar} neutral steroids, estrenediol and 19-nor-T. Isolation of 19-oic-A, estrenediol, and 19-nor-T in pig ovarian follicular fluid provided evidence that the metabolic pathways observed in culture were reflective of in vivo follicular steroidogenesis. The effects of metabolites of A-dione were tested for their ability to modulate parameters associated with follicular development and maturation. Granulosa cells isolated from prepubertal pigs were cultured with various combinations of FSH and steroids to assess the effects of these hormone combinations on the induction of LH receptors and aromatase activity. 19-nor-T significantly augmented the ability of FSH to induce LH receptors on granulosa cells, while estradiol and estrenediol were without effect. Likewise, the androgens A-dione, testosterone(T), 19-nor-T and all significantly enhanced the FSH stimulation of granulosa cell aromatase activity, while estradiol was without effect.;To determine if the regulation of LH receptor and aromatase induction by androgens, but not estrogens could be explained by the expression of steroid hormone receptors, immunohistochemical studies were performed to localize androgen and estrogen receptors in the pig ovary. Granulosa cells from small preantral and antral follicles expressed androgen receptors, but were devoid of estrogen receptors. As follicles increased in size as a result of follicular development and maturation, the expression of androgen receptors decreased. Weak expression of estrogen receptors was observed only in mature follicles and the developing corpus luteum. Collectively, these results were interpreted to indicate that androgens are potentially important modulators of FSH action during follicular development and maturation.

ATP utilization by P-type cation transport ATPases includes a phosphorylated intermediate which is formed by transfer of the ATP terminal phosphate onto an aspartyl residue at the catalytic site. The phosphorylation site and cation binding site of the ATPase molecule are separated by a fairly long distance of about 50 A. The coupling mechanism of these two functional sites is not yet fully understood and is currently under active investigation. Within the family of cation transport ATPases, the Ca2+-ATPase of sarcoplasmic reticulum (SR) provides an advantageous experimental system due to its abundance in the native membrane, and the availability of cDNA for expression of functional protein.;The sarcoplasmic reticulum Ca2+-ATPase segment extending from the phosphorylation site (Asp351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6 and M8), shares a marked sequence homology with the corresponding segments of other cation ATPases. We generated twenty six point mutations in this segment and expressed those mutant enzymes in COS-1 cells. We found that non-conservative mutations of residues which are homologous in various cation ATPases result in strong inhibition of catalytic and transport functions. Mutations of non-homologous residues to match the corresponding residues of other cation ATPases are not inhibitory, and in some cases produce higher activity. The inhibitory mutations specifically affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+, which is required for enzyme phosphorylation by ATP. This indicates that activation of the phosphoryl transfer reaction by Ca2+ binding, and vectorial displacement of bound Ca2+ by enzyme phosphorylation, do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece, through the M4 helix in the membrane bound region, sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.

Neuronal nicotinic acetylcholine receptors (nAChRs) in the brain, especially in the hippocampus, are thought to play a crucial role in the physiology of learning and memory. Characterization of the neuronal nAChRs by molecular, immunological and physiological techniques has revealed that their molecular structures, physiology and pharmacology are highly diverse and different from those of the muscle nAChR. However, little is known at this time about noncompetitive antagonism of these nAChRs. The purpose of this study was to characterize the interaction of bupivacaine, an open channel blocker of muscle nAChR, with the neuronal nAChRs in cultured hippocampal neurons. Using whole-cell and single-channel modes of the patch-clamp technique, the interactions of bupivacaine with the neuronal nAChRs were studied under different experimental conditions. Whole-cell currents with two different types of decay kinetics could be induced by ACh (300 muM). Currents that were fast-decaying were highly sensitive to blockade by the competitive nicotinic antagonist methyllycaconitine (MLA) at 10 nM. Currents having a slow decay were only partially blocked by MLA. Both the fast and slowly decaying ACh-induced whole-cell currents were blocked by bupivacaine, but with different sensitivity. Open-channel blockade by the drug was evidenced by a substantial decrease in the decay time constants of slowly decaying whole-cell currents and was confirmed by a shortening of the lifetime of ACh-activated single channels. The weak voltage dependence of the reduction of the decay time constant of the slowly decaying current suggests that the bupivacaine-binding site(s) responsible for open-channel blockade is(are) probably located near the orifice of the receptor ion channel. The non-linear relationship of this blockade with increasing concentration of bupivacaine indicated two distinct blocking rates, and suggested a closed-channel interaction of the drug at higher concentration, which was supported by the bupivacaine-induced decrease in the probability of a channel being opened. The blockade of the slowly decaying current by bupivacaine at high concentration was use dependent. Bupivacaine decreased the peak amplitude but did not change the decay time constant of the fast decaying ACh-induced currents, suggesting that a closed-channel blockade, but not open-channel blockade was taking place. This closed-channel blockade was time dependent and was noncompetitive. The neuronal nAChRs were more sensitive than NMDA, kainate, quisqualate, GABA, or glycine receptors to the action of bupivacaine.