Catecholamine content and adenylate cyclase activity in corpora lutea of different ages of the PMSG-treated immature rat

The catecholamine content and adenylate cyclase response were studied in a well-characterized corpus luteum model, where ovulation was induced by treatment of prepubertal Sprague-Dawley rats with pregnant mare's serum gonadotropin. The luteal content of noradrenaline, determined with HPLC, was constant during the first 7 days of pseudopregnancy, followed by a 3-fold increase in older corpora lutea. No detectable amounts of dopamine were found, while trace amounts of adrenaline were found in a few cases. The increase in noradrenaline content was not associated with a changed sensitivity of luteal adenylate cyclase to catecholamines. The response to adrenaline was maximal in 3-day-old corpora lutea, whereafter a decrease was seen. The significance of the increased endogenous levels of noradrenaline at the end of pseudopregnancy is at present unknown. However, the fact that the increase in noradrenaline occurs a few days before spontaneous luteolysis is of special interest, since it has been suggested that an adrenergic innervation is a prerequisite for the antigonadotropic effect of prostaglandin F2 alpha in the human corpus luteum.     

Evidence for rapid loss of spare hCG receptors in the corpora lutea of the hypophysectomized rat

In pregnant mare serum gonadotropin treated immature rats hypophysectomized on the day of ovulation (day 1) the corpora lutea (CL) persist as normal morphological structures and produce steroids, especially 20-dihydroprogesterone (20-DHP), for at least 40 days (Taya and Greenwald, 1982), although there is a rapid decline in human chorionic gonadotropin (hCG) binding sites (Kim and Greenwald, 1984). In this study the number of occupied and non-occupied hCG receptors in CL from hypophysectomized rats decreased to 14% and 36%, respectively, compared to intact day 5 pseudopregnant animals, but the binding affinity was unchanged. Decreased concentration of occupied hCG receptors paralleled hormonal levels of serum luteinizing hormone (LH) which were measurable in only 9 out of 20 animals and were near the lower limits of the assay (40 pg/ml). Luteal progesterone (P₄) production in response to LH was markedly decreased after hypophysectomy, but the maximal P₄ response to LH and 20-DHP production in response to LH and 8-Br-cAMP were the same in hypophysectomized and intact animals. Although hCG receptor concentration in CL decreased significantly after hypophysectomy, LH-stimulated luteal production of cAMP was almost the same in both groups. These results indicate that LH spare receptors which are uncoupled from cAMP exist in the CL of the intact pseudopregnant rat and that after hypophysectomy they quickly disappear within 4 days.     

Adenylate cyclase activity during development and reversal of cardiac hypertrophy

Inotropic responses to isoproterenol of hypertrophied hearts have been shown to be decreased. We have previously reported that in 13-week-old spontaneously hypertensive rats (SHR) this decrease is probably due to decreased beta-adrenergic receptor number, while in hearts from two kidney-one clip renal hypertensive rats (2K-1C RHR), this is due to a decreased nucleotide regulatory protein activity. We now show that changes in 2K-1C RHR are time dependent. One week after instituting development of hypertension the heart is already hypertrophied. Biochemical changes consistent with decreased glucagon receptors are seen, as well as beginning changes consistent with decreases in the nucleotide regulatory protein activity. By two weeks this is more evident. Hypertrophy and biochemical changes can be reversed up to six weeks, but by ten weeks the activity of the catalytic subunit of the adenylate cyclase system is decreased. In 1K-1C RHR, biochemical changes in the cyclase system are accelerated as compared with the 2K-1C model. In SHR, changes in 24-week-old rats are the same as in the 13-week-old rats. It is concluded that in cardiac hypertrophy associated with different models of hypertension the decreased inotropic responsiveness to isoproterenol is associated with different biochemical defects in the beta-adrenergic receptor response coupling pathway, and that reversal in function occurs only when there is no apparent change in the catalytic subunit of the adenylate cyclase complex.     

Alterations in the cellular membranes of regressing rat corpora lutea

Wide angle x-ray diffraction and fluorescence polarization were used to determine the structural properties of membranes from rat luteal cells. Examination of a plasma membrane fraction by x-ray diffraction revealed a significant increase in the gel phase melting temperature during luteal regression. The membrane fluidity of this fraction as well as that of a preparation of microsomes was also studied by fluorescence polarization. Using a fluorescent probe, membrane fluidity was observed to decrease during luteolysis. The temporal correlation between structural changes in the membrane and decreased progesterone secretion suggests that alterations in the physical properties of cellular membranes may be involved in the process of luteal cell regression.     

Progestational function of perifused rat corpora lutea.

Corpora lutea (CL) from synchronously ovulated, prepubertal rats remained viable in perifusing Eagle medium (Dulbecco modified) flowing at a rate of 1 ml/hr. As an index of viability, progesterone (P) and 20alpha-hydroxypreng-4-ene-3-one (20alpha-OH-P) were determined by radioimmunoa-say in effluent media. Corpora lutea removed on the 2nd day of diestrus (D-2) or the 2nd day of pseudopregnancy (PP-2) showed fatigue by a continuous decline in the concentrations of effluent progestins. P decreased from 2 to 0.2 ng/ml/CL during an interval of 5 hr, while 20alpha-OH-P decreased from 2.2 to about 0.7 ng/ml/CL. In contrast corpora lutea taken on PP-4 maintained higher progestin levels in the effluent media. P present initially at 2.2 ng/ml/CL decreased to 0.73 ng/ml/CL; 20alpha-OH-P, initial concentration 0.84 ng/ml/CL, decreased to 0.41 ng/ml/CL. The greater functionality of PP-4 CL indicated by the continued predominance of , suggested an in vivo imposition of a luteotropic stimulus occurring after PP-2. Addition of bovine LH (NIH-LH-B8, 20 mug/ml) to the perifusing medium stimulated steroidogenesis by PP-2 CL during which 20alpha-OH-P levels remained between 2.3 and 3.2 ng/ml/CL throughout perifusion, while P secretion decreased from 2.9 to 0.9 ng/ml/CL. Prolactin (NIH-P-B3, 20 mug/ml), did not significantly alter the original secretion pattern of PP-2 CL. During combined prolactin (20 mug/ml) and LH (2 to 10 mug/ml) perifusion, P secretion predominated, decreasing from 3.9 to 1.0 ng/ml/CL; 20alpha-OH-P decreased from 1.2 to 0.7 ng/ml/CL.     

Examination of the role of 'non-functional' corpora lutea in the female rat

In rats with 5 day reproductive cycles, 'anovulatory' cycles were induced by blockade of ovulation with sodium pentobarbitone injected at pro-oestrus. Such anovulatory cycles were characterized in the ovaries by gradual atresia of the large follicles present at the time of the injection and the growth of a new cohort destined for the next ovulation. In the vaginal smears, anovulatory cycles were indistinguishable from normal ovulatory cycles. The serum concentrations of progesterone remained at baseline levels during dioestrus of anovulatory cycles whereas increased concentrations of progesterone were observed during dioestrus of ovulatory cycles. It is concluded that the 'non-functional' corpora lutea of the cycle are the source of dioestrous progesterone. The length of anovulatory cycles after a single injection of pentobarbitone was 5 days despite the absence of any increase in progesterone concentrations during dioestrus. It is concluded that progesterone production during dioestrus plays no major role in the control of the duration of 5 day reproductive cycles.     

Progesterone production by ectopic corpora lutea is independent of luteinizing hormone-stimulated adenylyl cyclase during early luteal development in the rabbit

Although estradiol is luteotropic in rabbits, corpora lutea develop and secrete progesterone in the absence of estradiol for 4-5 days. This period of estradiol independence is associated with high levels of LH-activated adenylyl cyclase and low levels of estradiol receptor. To determine if progesterone secretion is dependent on LH-stimulated adenylyl cyclase during this period of development, ectopic corpora lutea were established in ovariectomized rabbits. Rabbits were injected daily or twice daily with saline or 50, 100, or 150 IU hCG to desensitize LH-responsive adenylyl cyclase. On day 3, 4, 5, or 7, adenylyl cyclase activity was measured in luteal homogenates, and serum progesterone levels were determined by RIA. Basal and LH-stimulated adenylyl cyclase levels rose through day 5 of pseudopregnancy. Daily treatment with 50 or 100 IU hCG did not alter basal activity, but decreased LH-stimulated adenylyl cyclase activity by approximately 50% on day 3, by 80-90% on day 4, and by 90-95% on days 5 and 7. Serum progesterone levels were not different in hCG- and saline-treated rabbits. When LH-stimulated adenylyl cyclase was completely desensitized on days 3 and 4 of pseudopregnancy with twice daily injections of 150 IU hCG, serum progesterone levels were again, not different in saline- and hCG-treated rabbits. These results demonstrate that progesterone production by ectopic corpora lutea is not altered when LH-stimulatable adenylyl cyclase is desensitized and suggest that this early period of luteal development is independent of LH as well as estradiol.     

Adenylate cyclase and guanylate cyclase activity in normal and leukemic human lymphocytes

Adenylate cyclase (AC) and guanylate cyclase (GC) activities were studied in normal B-enriched and T-enriched lymphocytes, in lymphocytes of children with acute lymphocytic leukemia (ALL), and in lymphocytes of adults with chronic lymphocytic leukemia (CLL). AC activity was greater in normal B than T lymphocytes (215 pmole/min/mg protein versus 80 pmole in the membrane-enriched fraction) and i both increased greatly after stimulation with isoproterenol and more so with prostaglandins E and F2 alpha. In leukemic lymphocytes, AC showed depressed activity (20 pmole in ALL cells and 55 pmole in CLL cells) and was less sensitive to hormonal stimulation: this loss of sensitivity occurred to a greater extent in ALL than in CLL lymphocytes. GC activity was greater in normal T than B cells (in membrane-enriched fraction: 10.2 pmole versus 5.3 pmole). It increased little with isoproterenol and prostaglandins stimulation, and much more with sodium azide and dehydroascorbic acid stimulation. GC activity was increased in both types of leukemic lymphocytes (23 pmole for ALL cells and 18 pmole for CLL cells) and was insensitive to stimulation. Possible derangement of cyclase and cyclic nucleotide regulation in leukemic cells is suggested.     

Effects of oestradiol on steroid dehydrogenase activities in rat corpora lutea

The activities of 20alpha-hydroxysteroid dehydrogenase and 3beta-hydroxysteroid dehydrogenase in rat corpora lutea during the second half of pregnancy were measured. In luteal tissue of the intact pregnant rat, 20alpha-hydroxysteroid dehydrogenase activity was undetectable between days 12 and 18 of pregnancy but appeared slowly after hypophysectomy and hysterectomy on day 12. Treatment of the hypophysectomized and hysterectomized animal with oestradiol delayed this increase until day 17, at which time a rapid induction of this enzyme occurred. In the normal pregnant rat mean luteal 3beta-hydroxysteroid dehydrogenase activity increased between days 12 and 18 (P less than 0.05, Student's t-test) but fell rapidly after hypophysectomy and hysterectomy on day 12. Oestradiol treatment prevented this fall in activity and enzyme activity was not distinguishable from that of the intact rat. Progesterone secretion correlated well with the activities of these two enzymes in the three conditions examined.     

Adenylate cyclase activity in human pancreatic adenocarcinoma cell lines

Summary Conclusion BxPC-3, Hs 766T, Capan-2, Panc-1, and Capan-1 cells possess receptors for VIP and β-adrenergic agonists that are functionally coupled to adenylate cyclase. In this respect, they resemble pancreatic duct cells. However, we speculate that the process of neoplastic transformation has either downregulated the expression of secretin receptors or led to a defect in the receptor itself, placing a question mark over the usefulness of these adenocarcinoma cell lines as models of the pancreatic ductal epithelium. Background Because of the importance of ducts in pancreatic disease, we wished to establish which duct cells receptors are functional on adenocarcinoma cell lines. Methods We investigated the expression of agonist-stimulated adenylate cyclase activity in six human pancreatic adenocarcinoma cell lines. Known stimulants of pancreatic ductal secretion, VIP, PHI, secretin, β-adrenergic, and dopamine, were tested. Results For responsive cell lines, VIP was the most effective stimulant followed by adrenaline, isoprenaline, PHI, and secretin. Dopamine was without effect. Since high concentrations of PHI and secretin were required to stimulate cyclase activity, their effect is probably mediated by VIP receptors. Based on the degree of stimulation observed with the individual agonists, Hs 766T and BxPC-3 were the most responsive cell lines, followed by Capan-2 and Capan-1, and finally Panc-1. MIAPaCa-2 cells did not respond to any of the agonists tested.     

Adenylate cyclase activity in the epididymal adipose tissue from obese-hyperglycaemic mice

Summary The adenylate cyclase activities in 1,500 × g to 25,000 × g sediments of adipose tissue from 3 month old C57BL 6J/ob and lean mice were compared. Three anomalies were observed in obese mice: 1) a defect in basal (–80%) and fluoridestimulated (–50%) activities suggesting a decreased number of catalytic units per mg protein; 2) an impairment in stimulation by isoproterenol (–85%) with no change in apparent affinity for the hormone; and, 3) no stimulatory effect of ethanol upon guanyl nucleotide-stimulated activity. The two latter effects may be due to a reduced number of-adrenergic receptors per mg protein and/or to an alteration in the transducing process. On the other hand, guanyl nucleotides as well as prostaglandin PGE₁ exerted similar effects in the preparations from both lean and obese adipose tissue.List of abbreviations ob/ob obese-hyperglycaemic - cyclic AMP adenosine 3,5-cyclic monophosphate - Gpp(NH)p guanyl-5-yl imidodiphosphate - PGE₁ prostaglandin E₁     

Evidence for a dual source of relaxin in the pregnant rat: immunolocalization in the corpora lutea and endometrium.

Uterine tissue was collected from intact pregnant rats on days 4, 10-15, 18, and 22 of pregnancy and day 2 postpartum and from rats on day 22 of pregnancy after ovariectomy (day 9) and progesterone-estrogen treatment. Placental, cervical, mammary, and pituitary tissues were collected from intact pregnant rats on day 22. Ovarian tissue was collected from intact pregnant rats on days 15, 20, and 22. These tissues were evaluated for relaxin immunostaining at the light microscope level using an antiserum to purified rat relaxin and the avidin biotin immunostaining technique. Since the corpus luteum is the major source of relaxin in the rat, this tissue was used as a positive control. Relaxin immunostaining in the corpus luteum was observed in a discrete region of the cytoplasm. This pattern of staining corresponded with the discrete clustering of relaxin-containing secretory granules found in the rat luteal cells. Relaxin was not observed in the day 22 placenta, cervix, mammary gland, pituitary gland, or day 4-13 pregnant uterus. Relaxin immunostaining was detected in endometrial epithelial cells on days 14-22 of pregnancy and day 2 postpartum. Relaxin immunostaining was more evident at implantation sites than between implantation sites. Antimesometrial epithelial cells at implantation sites contained relaxin immunostaining. The mesometrial surface, which consists of the placenta and decidualized endometrium, did not contain relaxin immunostaining. Relaxin immunostaining was also present in the endometrial epithelial cells of the day 22 pregnant rats that had been ovariectomized on day 9 and given subsequent replacement therapy with ovarian steroids. The presence of relaxin immunostaining in the ovariectomized rat indicates the endometrium is not sequestering relaxin secreted from the corpus luteum. These data provide evidence that the pregnant rat endometrium is a source of relaxin in addition to the corpus luteum. The appearance of relaxin in endometrial epithelial cells after implantation and its prominence at implantation sites may indicate that locally secreted conceptus factors stimulate endometrial relaxin synthesis.