Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Alterations in uterine and placental prostaglandin F and E with gestational age in the rat

Experiments were designed to determine the chronological alterations in placental and uterine prostaglandin F and E (PGF and PGE) during pregnancy in the rat. Pregnant rats (sperm in the vagina = day 0) were sacrified at days 15, 18,19, 20, 21 and delivery (day 21 ) and placental and uterine tissues assayed (RIA) for PGF and PGE immediately (“ ”) or after 1 hour incubation (“ ”). Uterine content of PGF and PGE (ng PG/mg DNA) was increased significantly by day 19 and further increases were seen through delivery. Incubation of uterine tissue resulted in enhanced net production of PGF and PGE (p <.05) per mg DNA (as judged by tissue content and release into the incubation medium) by day 18 of pregnancy vs. day 15. Net production peaked around the time of delivery thus paralleling the alterations in tissue content .By contrast, no differences with gestational age were found in placental content of PGF and PGE , the concentrations throughout late gestation remaining in the range of uterine PGs at day 15. However, production of PGs per mg placental DNA increased markedly during incubation with significant enhancement detected by day 19 vs. 15, achieving levels even greater than the uterus .The and findings for the uterus are consistent with the hypothesis that increases in uterine PGs levels at the end of pregnancy may play an important role in parturition. The experiences with placental tissue suggest that the potential for PG production per placental cell may also increase in late gestation and thereby contribute to the augmented intrauterine availability of PGs at that time.     

Retinopathy of prematurity (ROP) and indomethacin therapy in premature infants with paten ductus arterious (PDA)

Recent studies suggested that prostaglandins (PGs) may play a role in the pathogenesis of retinopathy of prematuriry (ROP). To evaluate if PGs inhibitor, indomethacin, would affect the incidence or severity of the ble-blind controlled study of indomethacin for the closure of PDA. Twenty-three were in the control group and 24 in the indometchin group. Indirect ophthalmospic examinations wee performed from about 4 weeks of postnatal age and onward and needed. There was no significant differences between the groups with respect to birth weight, gestational age, postnatal age, Apgar score, and cardiopulmonary status shortly after birth and at the time of study.Six in the control and 2 in the indomethacin group (p=0.58) developed active ROP; one in each group developed cicatricial ROP. It appears that with current doses of therapy, indomethacin does not increase the incidence or severity of ROP.     

Effects of indomethacin on intestinal secretion, prostaglandin E and cyclic AMP: Evidence against a role for prostaglandins in cholera toxin-induced secretion

The effects of indomethacin on intestine mucosal cAMP, intestinal fluid secretion, and mucosal and fluid PGE were studied in rabbits in vivo following challenge with cholera toxin. Indomethacin had no effect on cholera toxin-induced fluid secretion or cAMP accumulation. Inhibition of PGE synthesis was achieved by the administration of two but not one injection of indomethacin. These studies provide evidence against a role for PGE in mediating cholera toxin-induced secretion and point out the need to measure prostaglandin levels when using prostaglandin synthetase inhibitors in vivo.     

Selective phosphorylation of myosin light chain in intact skeletal muscle

The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [³²P]phosphate per mole of light chain in ³²P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle.     

Inhibition of in vitro human chorionic gonadotropin-stimulated testosterone production in testis and of ovulation in the rat by charcoal-treated rat testicular extract

Previously, we described the presence of a factor obtained from a 105,000 X g supernatant of rat testis that was found to inhibit human chorionic gonadotropin (hCG) binding to gonadal receptors. In the present study, similarly prepared testicular extract was tested for its effects on in vitro hCG-stimulated testosterone production by isolated testis interstitial cells and for its effect on spontaneous ovulation in the rat. Incubation of interstitial cells with charcoal-treated extract significantly inhibited the steroidogenic response to hCG in a dose-related manner. This inhibition was also apparent after heating the extract for 10 min at 100 degrees C. Preincubation of the cells with charcoal-treated extract resulted in an inhibitory effect that was not readily reversed by subsequent addition of hCG, revealing an element of irreversibility in the mechanism of inhibition. A single i.p. injection of testicular extract given between 1430-1630 h of proestrus inhibited spontaneous ovulation in the rat. This effect was also observed after heating the extract for 10 min at 100 degrees C; in contrast, no significant effect was obtained with the injection of a similar dose of liver extract. Administration of 5 IU hCG after pretreatment with the testicular extract did not reverse the inhibitory effect on ovulation, indicating that this effect was probably not exerted at the hypothalamus-pituitary level. It is concluded that the aqueous testicular extract contains a factor able to antagonize the physiological events mediated by luteinizing hormone (LH)/hCG, and that this factor is consistent with the presence of an LH/hCG-binding inhibitory activity in rat testis.     

Allelic allotype inclusion and exclusion among rabbit Ig-bearing lymphocytes of peripheral blood and lymphoid organs

Mononuclear cells isolated from peripheral blood, appendix, sacculus rotundus, mesenteric lymph nodes, and spleen of b⁴b⁵ heterozygous rabbits were examined for surface Ig allotypes of the b locus. Ig allotype-bearing cells were detected as cells binding erythrocytes or bacteria coated with monospecific anti-b4 or anti-b5 antibody (Ab). Rosetting the cells with Ab-coated erythrocytes indicated that many peripheral blood lymphocytes, but relatively few appendix cells, bore both the b4 and b5 allotypes. Lymphocytes bearing both the b4 and b5 allotypes were also detected by incubating the cells with a mixture of Escherichia coli coated with anti-b4 Ab and Gaffkya tetragena coated with anti-b5 Ab. The percentage of Ig-positive lymphocytes binding both bacteria was 22–31% in the peripheral blood, 4–6% in the appendix, 3–5% in the sacculus rotundus, 4–10% in the mesenteric lymph nodes, and 5% in the spleen. Thus, the percentage of double-bearing lymphocytes was higher in the blood than in the appendix, sacculus rotundus, mesenteric lymph nodes, or spleen. The b4b5-bearing cells in the blood were not cells with adsorbed cytophilic Ab, since these cells still bore both the b4 and b5 allotypes after pronase digestion and Ig regeneration. These double-bearing lymphocytes, i.e., cells exhibiting allelic allotype inclusion, are probably less differentiated cells.     

The effect of beta-endorphin on natural cytotoxicity and antibody dependent cellular cytotoxicity

The ability of the central nervous system to modulate immune responsiveness has received increasing attention. A potential mechanism that would allow the central nervous system to alter the immune system is the release of neuroendocrine and neurotransmitter polypeptides into the peripheral circulation with subsequent modulation of immunocyte function. In this report, we demonstrate that the neuropeptide, beta-[D-ALA2]-endorphin augments natural cytotoxicity but does not effect antibody-dependent cellular cytotoxicity. The observations are discussed in relation to the mechanisms for natural cytotoxicity and antibody dependent cellular cytotoxicity.     

Modulation of progestin secretion in ovarian cells by 17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone): a direct demonstration in monolayer culture.

These studies with a monolayer system of porcine granulosa cells provide a direct demonstration of the ability of androgen to stimulate progestin secretion by ovarian cells. A preferential action of the more potent androgens, dihydrotestosterone and testosterone, was shown but only dihydrotestosterone demonstrated the capacity to stimulate progestin secretion throughout the culture period. Estradiol 17-β markedly depressed progestin synthesis. The results suggest a modulatory role for androgens in the development of full steroidogenic potential by ovarian granulosa cells during follicular development.     

Effect of the prostacyclin synthetase inhibitor tranylcypromine on uterine blood flow in pregnancy

Prostacyclin is a potent vasodilator in a number of vascular beds including the uterus. However, the role of prostacyclin in maintaining uterine blood flow during pregnancy is not well established. Recent reports have appeared suggesting that tranylcypromine can selectively inhibit prostacyclin synthesis. Thus, the present study was undertaken using an unanesthetized chronically catheterized pregnant sheep preparation to evaluate the effects of direct intra-arterial infusions of tranylcypromine on the uterine vasculature of late-term pregnant ewes. Infusions of 1, 3 and 10 mg/min of tranylcypromine led to dose-related reduction in uterine blood flow (16, 21 and 47 percent, respectively) and increased blood pressure (7, 10 and 23 percent, respectively). However, these alterations were not associated with reductions in the uterine production rates of the prostacyclin metabolite, 6-keto-PGF_1α, as determined by unextracted plasma RIA. In addition, pre-treatment of animals with the α-adrenergic blocking agent, phenoxybenzamine, almost totally abolished uterine and systemic blood pressure responses to tranylcypromine. These data suggest that tranylcypromine either releases or elevates levels of an alpha adrenergic stimulant which constricts the uterine and systemic vasculature and does not alter prostacyclin levels at the dose tested.     

Reduced muscarinic receptor plasticity in frontal cortex of aged rats after chronic administration of cholinergic drugs

Age-related differences in muscarinic receptor plasticity were observed in young, adult and senescent Fischer 344 rats (3, 9 and 27 months old, respectively) following the chronic, intracerebroventricular (ivt) administration of a cholinergic agonist, oxotremorine, or antagonist, methylatropine. After three weeks treatment of young rats with ivt oxotremorine, the maximum number (Bmax) of 3H-QNB binding sites in frontal cortex, determined by saturation experiments, was reduced by 27%, with no apparent change in the affinity (Kd) of 3H-QNB for the muscarinic receptor. Conversely, chronic ivt methylatropine administered to 3 month old animals caused a 29% increase in Bmax with no significant change in Kd. Adult animals showed a somewhat lesser degree of muscarinic receptor plasticity (16% down-regulation after oxotremorine, 22% up-regulation after methylatropine). However, 3H-QNB binding parameters in frontal cortex from senescent rats were not significantly altered following identical treatments with oxotremorine or methylatropine. Thus, muscarinic receptor adaptation to chronic, cholinergic drug administration was impaired in aged animals. This reduced receptor plasticity with aging could have important implications for the long-term drug treatment of elderly patients and for the therapeutic efficacy of cholinergic drugs in age-related neurological disorders, such as Alzheimer's disease.     

Age-related changes in in vitro immunoglobulin secretion: Comparison of responses to T-dependent and T-independent polyclonal activators

In vitro Ig secretion by peripheral blood mononuclear cells (MNCs) from old and young donors, in response to T-dependent (TD) [pokeweed mitogen (PWM)] and T-independent (TI) [Salmonella paratyphii B (SPB)] activation were compared. In older donors, the IgG and IgA responses to PWM were comparable to those of young donors; the IgM response was reduced in the elderly. With SPB activation, IgA response was again preserved, whereas IgG response was reduced and IgM secretion was markedly decreased. These data indicate class-specific changes in Ig responsiveness to both TD and TI cell activators with age. The reduction in TI-induced IgG and IgM responses in the elderly suggest that changes in B cells themselves have occurred. The preservation of the TD IgG response in concert with reduced TI response indicates that a decline in T-suppressor influences over B cells in the elderly coupled with reduced B-cell synthesizing capacity can result in apparent “preservation” of the final Ig response. In keeping with the above postulate, analysis of individual elderly donors' responses indicated that some of the old donors responded to PWM, but not SPB; none of the old donors responded to SPB and not PWM. In contrast, some young donors did respond to SPB, but not PWM. These results also suggest that nonresponse to PWM in young donors relates to an override of functionally intact B cells by T-regulator influences.     

Identification of a rabbit B cell alloantigen with an antiserum produced by alloimmunization with thymus cells.

Alloimmunization with rabbit thymus cells resulted in an antiserum (anti-Rly) which was shown to react with rabbit lymphocytes by an indirect rosette technique. The titration curve obtained with dilutions of anti-Rly antiserum on lymph node cells revealed two plateaus indicating that the antiserum was multispecific; at low dilutions of antiserum, within the first plateau, both B and T cells were rosetted whereas at high dilutions, within the second plateau, only B cells were rosetted. The antigen detected at high dilution was designated LB-1 (lymphocyte B cell alloantigen 1). The evidence that the cells identified within the second plateau are B cells is as follows: 1) simultaneous enumeration of LB-1⁺ and Ig⁺ (B) cells by use of distinguishable erythrocytes (sheep and human) as indicator cells revealed that of the 53% rosettes observed, essentially all (51%) were mixed rosettes containing both erythrocytes whereas simultaneous enumeration of LB-1⁺ and T⁺ cells (identified by anti-T cell antiserum) showed essentially no mixed rosettes (less than 2%); 2) approximately 80% of purified Ig⁺ (B) cells were identified as LB-1⁺ cells whereas essentially no (< 1%) purified T cells could be detected as LB-1⁺; 3) the percentages of LB-1⁺ cells and Ig⁺ cells were both reciprocal to the precentages of T⁺ cells identified in various lymphoid organs except for bone marrow; 4) the removal of LB-1⁺ cells from spleen cells of rabbits immunized with sheep red blood cells resulted in a depletion (42–71%) of direct plaque forming cells (PFC). Since the percentages of bone marrow cells rosetted using anti-LB-1 antiserum (approximately 70%) was much greater than the percentage rosetted using anti-Ig (approximately 10%), it appears that the anti-LB-1 antiserum is not directed against an Ig allotype. The titration curves of the anti-Rly antiserum on peripheral blood lymphocytes of a large rabbit family suggested that the LB-1 antigen on B cells is an alloantigen probably inherited in simple Mendelian fashion. Adsorption studies indicated that the LB-1 antigen on B cells is not detectable on brain, liver, kidney or erythrocytes.     

Plasma relaxin concentrations in the pig during the periparturient period: Association with prolactin,estrogen and progesterone concentrations

Concentrations of relaxin, prolactin, unchromatographed estradiol 17_β (E_2β) and progesterone (P₄) were measured in serial samples of inferior vena caval blood, in three pigs, during late pregnancy, and parturition. Maximal relaxin concentrations occurred 60 to 24h before parturition, and ranged from 60 to 286ng/ml. Prolactin concentrations increased from 12.5ng/ml, 48 to 36 hours before parturition, to between 79 to 184ng/ml. At the time of the relaxin surge, E_2β levels were high, and P₄ concentrations were decreasing, thus raising the $\text{E}{}_{\mathrm{2\beta }}\text{P}{}_4$ ratio. A surge in prolactin concentrations followed upon a decline of P₄ to less than 10ng/ml, coinciding with the increase in relaxin concentrations in 2 gilts, and following the surge in relaxin in the third. Udder development occurred near the time of increased relaxin concentrations. ‘Milk let down’ followed maximal relaxin and prolactin concentrations in two gilts, and the increase in prolactin, rather than the increase in relaxin concentration, in the third.     

The possible role of intracellular receptors in the expression of narcotic antagonist precipitated abstinence

The antagonist potencies of methylnaloxone and naloxone were measured for morphine in the isolated guinea pig ileum by the pA₂ method and a potency ratio determined. A second potency ratio was determined by comparing the concentration of each antagonist required to produce abstinence-like contractures of ileal tissues from morphine-treated guinea pigs. If the latter response required penetration of the cell membrane lipid bilayer, one would expect methylnaloxone, a quaternary amine, to be significantly less potent than naloxone. Comparison of potency ratios for methylnaloxone and naloxone did not support this hypothesis.     

Turnover of lipid-bound arachidonate and biosynthesis of prostanoids in the endometrium and myometrium of the laying hen

In vitro incorporation of [3H]arachidonate into various lipid fractions of minced avian endometrium and myometrium has shown that (i) phospholipids and triacylglycerols are labeled preferentially, whereas labeling of cholesteryl esters is only marginal; (ii) acylation reactions with arachidonoyl residues are very rapid in these tissues; (iii) incorporation of arachidonate into phosphatidylcholine and phosphatidylethanolamine seems to be a one-step reaction, whereas the transfer of arachidonate to phosphatidylinositol and phosphatidylserine proceeds after a time lag, suggesting that intermediate reaction steps are involved; and (iv) under conditions where tissue differences in free arachidonate levels are diminished by adding radioinert arachidonate to the incubation medium, endometrium incorporates more than twice as much [3H]arachidonate/unit of phospholipids than does the myometrium, whereas there is no such tissue-specific difference in the labeling of triacylglycerols. Furthermore, after a 90-min incubation phosphatidylserine and phosphatidylinositol not only are labeled markedly higher than either phosphatidylcholine or phosphatidylethanolamine in both tissues but the tissue-specific difference is also the highest (2.4- to 2.8-fold in the case of these phospholipids). Prostanoid synthesis from [3H]arachidonate in a crude membrane preparation in vitro has demonstrated that the myometrium possesses a significantly higher prostanoid-synthesizing capacity than the endometrium. This difference is particularly apparent (about 2.5-fold) in the case of prostaglandin E2. The results suggest that the endometrium is endowed with a special capacity to respond to signals with a rapid alteration of the level of free arachidonic acid, whereas the myometrium has the specific capacity to amplify its own contractions by an increased production of prostaglandins.     

Sodium-dependent high-affinity binding of [3H]hemicholinium-3 in the rat brain: a potentially selective marker for presynaptic cholinergic sites

This report describes the membrane binding properties of [3H]hemicholinium-3 ([3H]HC-3), a selective inhibitor of sodium-dependent high-affinity choline uptake (SDHACU) in cholinergic nerve terminals. Under the described assay conditions, [3H]HC-3 bind with a saturable population of high-affinity (apparent Kd = 1.9 nM) CNS membrane sites having the regional distribution: striatum much greater than hippocampus greater than cerebral cortex greater than cerebellum. High-affinity [3H]HC-3 binding is entirely dependent upon the presence of sodium chloride (EC50 = 35-50 mM) and is markedly reduced when other salts of sodium or monovalent ions are substituted. [3H]HC-3 binding is inhibited by choline (Ki = 6 microM) and acetylcholine (Ki = 35 microM) but markedly less sensitive to other cholinergic agents and metabolic inhibitors. In light of the similar ionic dependencies, regional distributions and pharmacological specificities of [3H]HC-3 binding and SDHACU, closely associated sites may be involved in both processes.