Support for a physiological role of endogenous catecholamines in the stimulation of bovine luteal progesterone production

To determine if catecholamines were present in bovine luteal tissue, corpora lutea (CL) were obtained during the mid-luteal phase (Days 10-12) and the concentration of dopamine (DA) and norepinephrine (NE) was determined by high-performance liquid chromatography. Both DA and NE were detected in luteal tissue at mean concentrations of 41.9 +/- 5.73 and 10.2 +/- 2.51 ng/g for DA and NE, respectively. These concentrations represented a luteal content of 306.6 +/- 66.88 ng/CL for DA and 70.5 +/- 16.88 ng/CL for NE. In vitro, DA at concentrations of 1.0 mM to 0.01 mM stimulated the production of progesterone (P4, p less than 0.05). The response to DA was inhibited by propranolol (a beta-adrenergic receptor antagonist, p less than 0.05) but not by phentolamine, phenoxybenzamine (alpha-adrenergic receptor antagonists), or haloperidol (a DA receptor antagonist, p greater than 0.05). Neither L-tyrosine nor L-dopa altered P4 production (p greater than 0.05). Inhibition of DA beta-hydroxylase, the enzyme that catalyzes the conversion of DA to NE by FLA-63 blocked the DA-induced increases in luteal P4 production (p less than 0.05). These results demonstrate the existence of DA and NE in bovine luteal tissue and indicate that exogenous DA can be converted to NE in luteal tissue. The results support a physiological role for catecholamines in the stimulation of bovine luteal function.     

Intraluteal infusions of prostaglandins of the E, D, I, and A series prevent PGF2 alpha-induced, but not spontaneous, luteal regression in rhesus monkeys

A luteotropic role for prostaglandins (PGs) during the luteal phase of the menstrual cycle of rhesus monkeys was suggested by the observation that intraluteal infusion of a PG synthesis inhibitor caused premature luteolysis. This study was designed to identify PGs that promote luteal function in primates. First, the effects of various PGs on progesterone (P) production by macaque luteal cells were examined in vitro. Collagenase-dispersed luteal cells from midluteal phase of the menstrual cycle (Day 6-7 after the estimated surge of LH, n = 3) were incubated with 0-5,000 ng/ml PGE2, PGD, 6 beta PGI1 (a stable analogue of PGI2), PGA2, or PGF2 alpha alone or with hCG (100 ng/ml). PGE2, PGD2, and 6 beta PGI1 alone stimulated (p less than 0.05) P production to a similar extent (2- to 3-fold over basal) as hCG alone, whereas PGA2 and PGF2 alpha alone had no effect on P production. Stimulation (p less than 0.05) of P synthesis by PGE2, PGD2, and 6 beta PGI1 in combination with hCG was similar to that of hCG alone. Whereas PGA2 inhibited gonadotropin-induced P production (p less than 0.05), that in the presence of PGF2 alpha plus hCG tended (p = 0.05) to remain elevated. Second, the effects of various PGs on P production during chronic infusion into the CL were studied in vivo. Saline with or without 0.1% BSA (n = 12), PGE2 (300 ng/h; n = 4), PGD2 (300 ng/h; n = 4), 6 beta PGI1 (500 ng/h; n = 3), PGA2 (300 ng/h; n = 4), or PGF2 alpha (10 ng/h; n = 8) was infused via osmotic minipump beginning at midluteal phase (Days 5-8 after the estimated LH surge) until menses. In addition, the same dose of PGE, PGD, PGI, or PGA was infused in combination with PGF2 alpha (n = 3-4/group) for 7 days. P levels over 5 days preceding treatment were not different among groups. In 5 of 8 monkeys receiving PGF2 alpha alone, P declined to less than 0.5 ng/ml within 72 h after initiation of infusion and was lower (p less than 0.05) than controls. The length of the luteal phase in PGF2 alpha-infused monkeys was shortened (12.3 +/- 0.9 days; mean +/- SEM, n = 8; p less than 0.05) compared to controls (15.8 +/- 0.5). Intraluteal infusion of PGE, PGD, PGI, or PGA alone did not affect patterns of circulating P or luteal phase length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Progesterone production by luteal cells isolated from cynomolgus monkeys: effects of gonadotropin and prolactin during acute incubation and cell culture

Corpus luteum function in cynomolgus monkeys (Macaca fascicularis) during the menstrual cycle and immediately following parturition was evaluated through in vitro studies on progesterone production by dispersed luteal cells in the presence and absence of human chorionic gonadotropin (hCG) or human prolactin (hPRL). Luteal cells isolated between days 17-20 of the menstrual cycle secreted progesterone (P) during short-term incubation (21.6 +/- 1.2 ngP/ml/5 X 10(4) cells/3 hr, X +/- S.E., n = 7) and responded to the addition of 1-100 ng hCG with a significant (p less than 0.05) increase in P secretion. Cells removed the day of delivery secreted large, but variable (27.9-222 ng/ml, n = 4) amounts of P during short-term incubation. Moreover, hCG (100 ng/ml) stimulation of P production by cells at delivery (176 +/- 19% of control) was less than that of cells from the cycle of (336 +/- 65%). The presence of hPRL (2.5-5000 ng/ml) failed to influence P secretion by luteal cells during short-term incubation in the presence or absence of hCG. P production by luteal cells obtained following delivery declined markedly during 8 days of culture in Ham's F10 medium: 10% fetal calf serum. Continual exposure to 100 ng/ml of hCG or hPRL failed to influence P secretion through Day 2 of culture. Thereafter hCG progressively enhanced (p less than 0.05) P secretion to 613% of control levels at Day 8 of culture. In contrast, hPRL significantly increased P secretion (163% of control levels, p less than 0.05) between Day 2-4 of culture, but the stimulatory effect diminished thereafter. The data indicate that dispersed luteal cells from the cynomolgus monkey provide a suitable model for in vitro studies on the primate corpus luteum during the menstrual cycle, pregnancy, and the puerperium, including further investigation of the possible roles of gonadotropin and PRL in the regulation of luteal function in primates.     

Serum androstenedione and testosterone concentrations during pregnancy and nonpregnant cycles in dogs

Concentrations of testosterone and of androstenedione were determined by radioimmunoassay in serum samples collected every 2-5 days throughout the periovulatory and luteal phases of the ovarian cycles of pregnant and nonpregnant beagle bitches. Testosterone levels were consistently lower than those of androstenedione, reached peaks of 29 +/- 4 ng/dl near the time of the preovulatory luteinizing hormone peak, and were reduced to near the limits of detection (less than or equal to 5-10 ng/dl) throughout the luteal phase. Androstenedione levels reached preovulatory peaks of 73 +/- 13 ng/dl, were 54 +/- 7 ng/ml during early estrus, increased (P less than 0.05) to early luteal phase peaks of 76 +/- 8 ng/dl between Days 6 and 18, and then declined to 41 +/- 5 ng/dl by Day 35-40 in both pregnant (n = 8) and nonpregnant (n = 4) bitches. Subsequent protracted increases in androstenedione occurred in 4 of 8 pregnancies but in none of the nonpregnant bitches. From Days 42 to 64 the differences in mean levels between pregnant (45 +/- 2 ng/ml) and nonpregnant (32 +/- 3 ng/ml) bitches was not significant (P greater than 0.05). At parturition androstenedione levels fell (P less than 0.05) abruptly from 39 +/- 7 to 13 +/- 3 ng/dl. These results suggest that, in the bitch, androstenedione is the major circulating androgen during the follicular and luteal phases and that patterns of androstenedione levels during the luteal phase parallel those reported for progesterone in pregnant and nonpregnant bitches, including maintenance of elevated levels throughout gestation and an abrupt decline at parturition.     

Functional differences between small and large luteal cells of the late-pregnant vs. nonpregnant cow

This paper describes an in vitro model for the study of two types of steroidogenic luteal cells from cows in different physiological states. Two different populations of enzymatically dispersed bovine luteal cells were separated on the basis of size in a Cel-Sep Sedimentation Chamber. The separated small (12.5-23 micron in diameter) and large (greater than 23 micron in diameter) luteal cells of late-pregnant cows (Days 190-280) contained the distinct morphological characteristics previously defined for these two populations of cells. Cells were evaluated for progesterone (P4) production during a 3-h incubation with and without bovine luteinizing hormone (bLH, 10 ng/ml). Both small and large luteal cells from the late-pregnant cow were found to contain equal levels of P4 at Time 0 and increased but equal levels of P4 after a 3-h incubation. Neither cell type showed an increase in P4 production in response to the addition of bLH (p greater than 0.05). Since these results differed from earlier reports for luteal cells of the nonpregnant cow, small and large luteal cells of the mid-cycle (Day 14) were incubated, and the levels of P4 production were compared with P4 levels from the late pregnant cow. In agreement with previous reports for nonpregnant cows, progesterone content at Time 0 was 7-fold higher in large cells than in small cells (p less than 0.05), and after 3 h of incubation, 13-fold higher (p less than 0.05). Although the small cells responded to the presence of bLH in the incubation medium with a 4-fold increase in P4 production, this increase was not significant (p greater than 0.05). The large cell did not respond to bLH. However, the large cell type continued to contain and produce more P4 than did the small cells treated with bLH. This study indicates that both the small and large luteal cells of late-pregnancy are able to produce P4. However, the large luteal cell of the estrous cycle produces greater quantities of P4 than does the small luteal cell or the large luteal cell of late pregnancy.     

Relaxin, oxytocin, and prostaglandin effects on progesterone secretion from bovine luteal cells during different stages of gestation

To determine the effects of relaxin, oxytocin, and prostaglandin F2 alpha on progesterone secretion, bovine luteal cells from different stages of gestation were dispersed in Medium 199 with 200 units/ml penicillin, 1.0% kanamycin, 0.5% bovine serum albumin, and 400 units/ml collagenase. Cells (10(5) were cultured in 400 microliters of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing fetal bovine serum and antibiotics, in Falcon multiwell plates, in a humidified environment of 95% O2 and 5% CO2 at 37 degrees C. Cells were cultured for 24 hr without treatment and thereafter with medium-hormone replacement every 24 hr. Progesterone was quantified from unextracted media by radioimmunoassay. Basal progesterone secretion after 24 hr was 1.81 +/- 0.14, 1.76 +/- 0.17, 0.54 +/- 0.49, and 0.57 +/- 0.21 pg/ml per viable luteal cell from 145-, 165-, 185-, and 240-day-old corpora lutea, respectively. Basal progesterone secretion increased (P less than 0.05) with time in culture. Relaxin induced a dose-dependent (greater than 100 ng/ml) increase in progesterone release, compared with the controls. Oxytocin and prostaglandin F2 alpha induced greater release (P less than 0.05) of progesterone than relaxin at all stages of gestation, but progesterone release was dependent on the stage of gestation and the duration in culture. Luteinizing hormone (100 ng/ml) stimulated whereas 17 beta-estradiol (50 ng/ml) inhibited progesterone secretion by luteal cells at all stages of gestation examined. Relaxin obliterated the prostaglandin- and oxytocin-induced progesterone secretion by bovine luteal cells from 145 to 214 days of gestation. Thus, relaxin, cloprostenol, and oxytocin regulate progesterone production by cultured bovine luteal cells, but hormone secretion was dependent on the stage of gestation.     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

PAF-induced contraction of canine trachea mediated by 5-hydroxytryptamine in vivo

We studied the secretory correlates of tracheal smooth muscle contraction caused by platelet-activating factor (PAF) in nine mongrel dogs in vivo. In five dogs, dose-response curves were generated by rapid intra-arterial injection of 10(-10) to 10(-6) mol PAF into the isolated tracheal circulation; tracheal contractile response was measured isometrically in situ. To examine the mechanism by which PAF elicits contraction of canine trachealis, concentrations of serotonin (5-HT) and histamine were assayed in the venous effluent as the arteriovenous difference (AVd) in mediator concentration across the airway for each level of contraction. PAF caused dose-related active tracheal tension to a maximum of 37.2 +/- 5.4 g/cm (10(-6) mol PAF). The AVd in 5-HT increased linearly from 0.20 +/- 0.05 (10(-9) mol PAF) to 3.5 +/- 0.3 ng/ml (10(-6) mol PAF) (P less than 0.005). In contrast, the AVd in histamine was insignificant and did not change with increasing doses of PAF. A positive correlation was obtained between the AVd in 5-HT and active tracheal tension (r = 0.92, P less than 0.001); there was no correlation between AVd in histamine and active tension (r = -0.16). PAF-induced parasympathetic activation was not mediated by 5-HT; contraction elicited by exogenous 5-HT was not affected by muscarinic blockade. We conclude that nonparasympathetically mediated contraction elicited acutely by PAF in dogs results at least in part from secondary release of serotonin and is not mediated by histamine.     

Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.     

A role for alternative pathway catecholamines in the regulation of steroidogenesis in cow luteal cells

Incubation of bovine luteal cells with the alternative pathway catecholamines octopamine, synephrine and deoxyadrenaline at concentrations of 10(-6) to 10(-3) M enhanced the production of progesterone (P less than 0.05). Tryamine did not alter basal progesterone production (P greater than 0.05). Addition of noradrenaline and adrenaline at concentrations of 10(-4) to 10(-7) M significantly elevated the production of progesterone (P less than 0.05). The steroidogenic response to noradrenaline and adrenaline was greater than that for octopamine, synephrine and deoxyadrenaline (P less than 0.05). Response to both primary (10(-6) M) and alternative (10(-4) M) pathway catecholamines was inhibited by propranolol (10(-5) M, P less than 0.05) but not phentolamine (10(-5) M, P greater than 0.05). These results demonstrate that octopamine, synephrine and deoxyadrenaline can affect steroidogenesis by bovine luteal cells, and their action is mediated by beta-adrenergic receptors.     

Effects of in vivo and in vitro administration of prostaglandin F2 alpha on lipoprotein utilization in cultured bovine luteal cells

Two experiments were conducted to determine if a loss in the ability to utilize lipoprotein-cholesterol is one mechanism whereby prostaglandin F2 alpha (PGF2 alpha) decreases steroidogenesis in bovine luteal cells. In the first experiment, serum-free cultures of bovine luteal cells were treated with PGF2 alpha (100 ng/ml) for 5 days prior to addition of lipoproteins. Exposure to PGF2 alpha completely suppressed low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-stimulated progesterone production (p less than 0.01) compared to control (no PGF2 alpha) cultures. Luteal cells cultured in the presence of LDL + luteinizing hormone (LH, 10 ng/ml) + PGF2 alpha produced significantly less progesterone than luteal cells cultured with LDL + LH (p less than 0.05). Treatment with PGF2 alpha had no significant effect on HDL + LH-stimulated progesterone synthesis. In the second experiment, cows were injected with a luteolytic dose of PGF2 alpha (25 mg), and the corpora lutea were removed at 0 (no PG), 1, 4, or 12 h post-injection. Dissociated luteal cells were placed in culture for 7 days, either with or without LH (10 ng/ml), and lipoproteins were added on Days 5-7. LH stimulation of progesterone production was apparent in cultures obtained at 0 and 12 (p less than 0.05) but not 1 and 4 h post-PGF2 alpha. Addition of either LDL or HDL increased progesterone synthesis in all cultures, regardless of time following in vivo administration of PGF2 alpha. It is concluded that PGF2 alpha can inhibit bovine luteal cell utilization of either LDL or HDL in vitro. However, luteal cell utilization of lipoproteins in vitro is not adversely affected by in vivo exposure to PGF2 alpha, if collected within 12 h post-PGF2 alpha.     

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.     

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A modified HPLC technique for simultaneous measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cerebrospinal fluid, platelet and plasma.

A sensitive, reliable and simplified HPLC assay for simultaneous measurement of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in human cerebrospinal fluid (CSF), platelets and plasma is described. Perchloric acid is used for one step precipitation of proteins and extraction of 5-HT and 5-HIAA. Precision of the assay has been increased by calibration of the instrument using serotonin-free plasma spiked with known amount of standards and N-w-methyl-5-hydroxytryptamine as internal standard. Integration of the peaks and calculations are achieved by a preprogrammed data module using ratio method. As little as 20 pg/ml of serotonin in the deproteinated sample can be detected using this procedure. In a group of surgical patients, plasma 5-HT concentration is (Mean +/- S D) 3.4 +/- 2.7 ng/ml and that of platelet 748.3 +/- 448.3 ng/10(9) platelets. In CSF, 5-HT is found to be 3.3 +/- 3.4 ng/ml and 5-HIAA is 15.1 +/- 7.3 ng/ml. A good correlation (r = 0.648, p less than .0001) is observed between 5-HT and 5-HIAA in CSF.     

Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-alpha bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-alpha. RA synoviocytes were cultured with TNF-alpha (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-alpha and p55 and p75 soluble receptors were measured using ELISA. TNF-alpha induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 +/- 23.3 ng/ml versus 27.4 +/- 20.9 ng/ml; P = 0.05). This high circulating TNF-alpha bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 +/- 23.7 ng/ml versus 3.4 +/- 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 +/- 6.2 ng/ml versus 0.0 +/- 3.0 ng/ml; P < 0.05). Levels of circulating TNF-alpha bioactivity were predictive of clinical response to TNF-alpha inhibition, confirming a key role for TNF-alpha in these RA patients.     

Role of platelets in contraction of canine tracheal muscle elicited by PAF in vitro

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.     

Progesterone and prostaglandin production by primate luteal cells collected at various stages of the luteal phase: modulation by calcium ionophore

Prostaglandins (PG) are produced by the corpus luteum (CL) of the rhesus monkey and may be involved in luteal regulation. Intracellular calcium has also been implicated as a mediator of luteolysis in domestic and laboratory species; however, its role in primate luteal function has not been investigated. The objectives of this study were to characterize temporal changes in basal and stimulated luteal PG production by CL of rhesus monkeys, and to examine the effects of calcium ionophore (CaI) on basal and gonadotropin-stimulated progesterone (P) production by the CL. CL were collected at various times after the estimated day of the luteinizing hormone (LH) surge: 5 days (early luteal phase, n = 4), 8-10 days (mid-luteal phase, n = 8), and 12-14 days (late luteal phase, n = 5). Dispersed luteal cells were incubated in the absence and presence of CaI, or with human chorionic gonadotropin (hCG) plus CaI at 37 degrees C for 8 h. PG and P concentrations in the medium were measured by radioimmunoassay. PGE2 and 6-keto-PGF1 alpha production decreased (p less than 0.05) from early luteal phase to mid-luteal phase and remained lower (p less than 0.05) during late luteal phase for all treatment groups. PGF2 alpha production decreased (p less than 0.05) from early to mid-luteal phase and rebounded in late luteal phase to the same level (p greater than 0.05) found in early luteal phase. CaI stimulated (p less than 0.05) basal PG production. The degree of stimulation was similar throughout the luteal phase (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in patterns of luteinizing hormone secretion before and after the first ovulation in the postpartum mare

To determine whether luteinizing hormone (LH) secretion during the first estrous cycle postpartum is characterized by pulsatile release, circulating LH concentrations were measured in 8 postpartum mares, 4 of which had been treated with 150 mg progesterone and 10 mg estradiol daily for 20 days after foaling to delay ovulation. Blood samples were collected every 15 min for 8 h on 4 occasions: 3 times during the follicular phase (Days 2-4, 5-7, and 8-11 after either foaling or end of steroid treatment), and once during the luteal phase (Days 5-8 after ovulation). Ovulation occurred in 4 mares 13.2 +/- 0.6 days postpartum and in 3 of 4 mares 12.0 +/- 1.1 days post-treatment. Before ovulation, low-amplitude LH pulses (approximately 1 ng/ml) were observed in 3 mares; such LH pulses occurred irregularly (1-2/8 h) and were unrelated to mean circulating LH levels, which gradually increased from less than 1 ng/ml at foaling or end of steroid treatment to maximum levels (12.3 ng/ml) within 48 h after ovulation. In contrast, 1-3 high-amplitude LH pulses (3.7 +/- 0.7 ng/ml) were observed in 6 of 7 mares during an 8-h period of the luteal phase. The results suggest that in postpartum mares LH release is pulsatile during the luteal phase of the estrous cycle, whereas before ovulation LH pulses cannot be readily identified.