O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus.

Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry.

Changing adrenergic sensitivity of the caruncular arterial vasculature supplying the bovine placentome.

With the advancement of gestation, blood flow increases preferentially to the caruncular bed of the gravid uterus in association with a decreasing sensitivity of the uterus to the vasoconstrictive effects of circulating catecholamines. This study directly compared the sensitivity of the caruncular artery (CA) of the isolated bovine placentome to phenylephrine (PE), a specific alpha 1-adrenergic receptor (AR) agonist, with that to norepinephrine (NE) and epinephrine (E), both of which are alpha 1-, alpha 2-, and beta-AR agonists, at two stages of gestation (140 to 170 d, mid-pregnant; 210 to 270 d, late pregnant). The CA of each placentome was perfused with oxygenated Krebs Ringer solution into which PE, NE or E were administered; increases in intra-arterial pressure were recorded. Further, NE content and numbers of alpha 1- and alpha 2-AR in the CA, intercaruncular arteries (ICA) and uterine arteries (UA) were quantitated. The CA from mid-pregnant cows exhibited greater (P less than .05) contractile responses to NE and E than did the CA from late pregnant cows, whereas responsiveness to PE remained constant. No difference in NE content, alpha 1-AR or alpha 2-AR numbers were observed in the UA, ICA or CA between mid-pregnant or late pregnant cows. Alpha 1-AR numbers were similar in CA, ICA and UA. However, CA contained threefold greater alpha 2-AR numbers than either the ICA or UA (50.2 +/- 6.1 vs 14.6 +/- 1.6 and 14.8 +/- 2.4 fmol/mg protein, respectively; P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and mechanical changes of uterine arteries during pregnancy in the pig.

A dramatic 15-fold increase in uterine blood flow in pigs occurs during pregnancy in association with marked increases in uterine arterial (UA) diameter. This study was conducted to determine UA collagen and elastin content, diameter, alpha 1- and alpha 2-adrenergic receptor (AR) numbers, norepinephrine (NE) and in vitro reactivity to phenylephrine on d 0, 20, 50, 80 and 110 of pregnancy in the pig. Uterine arterial collagen content declined progressively throughout pregnancy (P less than .01), whereas the content of elastin remained constant from d 0 to d 80, then increased (P less than .05) from d 80 to d 110. The UA collagen to elastin ratio was correlated with UA diameter (r = -.69; P less than .01), which increased from 4.5 mm on d 0 to 9.0 mm on d 110. Uterine arterial alpha 1-AR numbers remained low and constant throughout pregnancy, consistent with its retained ability to contract in response to phenylephrine. Uterine arterial NE content declined (P less than .05) from d 20 to d 80 before increasing slightly to d 110. Uterine arterial alpha 2-AR numbers remained high from d 0 to d 80 before decreasing (P less than .05) to low values on d 110. These data are consistent with a reduced adrenergic neuronal control and increased elasticity of the UA during pregnancy in the pig.

Role of 4-hydroxylated estradiol in reducing Ca2+ uptake of uterine arterial smooth muscle cells through potential-sensitive channels.

Entry of ionic Ca2+ into the vascular smooth muscle cell for contraction is thought to be mediated by two major membrane channels. The first are designated as potential-sensitive channels (PSCs), which are opened by membrane depolarization, and the second, as receptor-operated channels (ROCs), which are activated by alpha 1-receptor-ligand interactions. This study was designed to determine the presence of these 2 distinct populations of Ca2+ entry channels in smooth muscle cells of the uterine arteries in pigs. This was studied by measuring the baseline tone and contractile properties of uterine arteries in in vitro perfusion studies, as well as their specific Ca2+ uptakes. These parameters showed markedly different sensitivities towards two smooth muscle inhibitors used in this study: D-600 and amrinone. D-600 specifically inhibits uptake of extracellular Ca2+ through PSCs, while amrinone specifically inhibits Ca2+ uptake through ROCs. By choosing an appropriate concentration of D-600 or amrinone, Ca2+ uptake and contractions of uterine arterial segments induced by high-K+ (PSC activator) and phenylephrine (ROC activator) could be selectively inhibited. Furthermore, it was demonstrated that the blockade of Ca2+ uptake by D-600 and amrinone was additive, excluding the interpretation of a common Ca2+ pathway with two separate mechanisms for opening it. It was also determined that 4-hydroxylated estradiol (4OH-E2), a compound known to increase uterine blood flow in pigs, decreased Ca2+ uptake through the PSCs and exhibited no effect on ROCs. The presence of separate Ca2+ pathways that can be activated independently by agonists may indicate a refined system for controlling uterine blood flow.

Interaction of 4-hydroxylated estradiol and potential-sensitive Ca2+ channels in altering uterine blood flow during the estrous cycle and early pregnancy in gilts.

This study was conducted to define further the role of catechol estrogens (CE) as intermediates in estrogen-stimulated uterine hyperemia. Previous studies from our laboratory strongly suggest that changes in uterine blood flow (UBF) result from alterations in uterine arterial tone (distensibility) and/or contractility (reactivity to alpha 1-adrenergic agonists). Tone changes appear to set the baseline rate of flow, whereas contractility changes result in short-term reductions in luminal diameter. Changes in uterine arterial tone and contractility result from alterations in Ca2+ uptake through potential-sensitive channels (PSCs) and receptor-operated channels (ROCs), respectively. Uterine and mesenteric arteries were removed from 6 gilts at estrus (Day 0), 9 gilts on Day 13 of gestation (high estrogen, high UBF), and 8 gilts on Day 13 of the estrous cycle (low estrogen, low UBF). Arterial measurements included initial tone (baseline perfusion pressure [BPP] to a constant intraluminal flow) and increased tone after exposure to KCl, the contractility in response to the alpha 1-agonist phenylephrine, and specific uptake of 45Ca before and after exposure to the CE 4-hydroxylated estradiol (4OH-E2). Contractility of uterine arteries from Day 13 nonpregnant (NP) and Day 13 pregnant (P) gilts to phenylephrine were similar and significantly greater (p less than 0.01) than contractility of vessels from estrous gilts. The BPP and responses of uterine arteries from Day 13 NP gilts to KCl were greater (p less than 0.05) than the BPP and responses of arteries from Day 13 P and estrous gilts, which were similar to each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical localization of tissue-bound oestradiol in rat paracervical ganglion.

The uterine paracervical ganglion (Frankenhauser's ganglion) contains the terminal neurons of the cholinergic sacral parasympathetic, the short adrenergic sympathetic and the peptidergic (vasoactive intestinal polypeptide-containing) nerves of the internal genitalia. Previous studies have shown that either the number of cells or transmitter content of each of these neuronal systems is altered by variations in steroid hormones. Furthermore, our recent study showed that some component of the rat paracervical ganglion was capable of metabolizing [3H]oestradiol to oestrone and the 2-OH and 4-OH forms of oestrone and oestradiol. The present study employs the peroxidase-anti-peroxidase immunohistochemical method to localize oestradiol in rat paracervical ganglia. Specific reaction product was identified in (1) cytoplasm and some nuclei of principal ganglion cells, (2) cytoplasm of large vacuolated ganglion cells, (3) cytoplasm of 'small intensely fluorescent' cells and (4) some nerve fibres in ganglia from animals in oestrus. The cytoplasm of principal neurons and some nerve fibres exhibited specific staining for oestradiol in dioestrus and pro-oestrus. No oestradiol was localized in ganglia excised from animals in metoestrus. Preincubation in oestradiol before fixation was necessary for specific localization of oestradiol; treatment of tissues with oestradiol after fixation was not required. These results are not consistent with binding of oestradiol to the classical oestrogen receptor. The resistance of oestradiol to organic solvent extraction suggests that oestradiol is covalently bound to tissue proteins. Such covalently bound oestradiol has been reported as a by-product of tissue metabolism of oestradiol via P-450 enzymes.

A technique for monitoring blood flow changes with miniaturized Doppler flow probes.

A method for constructing miniaturized Doppler blood flow probes is presented. Since these probes weigh less than 100 mg and have crystal heads less than 0.5 mm in size, they are suitable for chronic placement on vessels as small as 200 micron. The probes are positioned under the vessel and rotated to optimize the Doppler signal. While held in that position, the crystal head is attached to the adventitia of the vessel with cyanoacrylate glue. A cuff holding the vessel and probe in the chosen position is then formed in situ by the application of a drop of silicone polymer. Data are presented showing the linearity of a flow probe response with the volume blood flow at rates as low as 14 microliter/min. With the use of the uterine artery of the conscious, unrestrained rat as an example, the probe was demonstrated to detect a dynamic change in flow.

Interaction of ovarian steroids and periarterial alpha 1-adrenergic receptors in altering uterine blood flow during the estrous cycle of gilts.

In vivo exposure to progesterone increases while estrogen decreases in vitro contractility of uterine arteries to nerve stimulation. In addition, uterine blood flow is highly correlated with the estrogen: progesterone ratio in systemic blood throughout the porcine estrous cycle (approximately 21 days). This study was conducted to compare the function of uterine periarterial sympathetic nerves of eight pigs during the follicular phase, the period of highest uterine blood flow and estrogen: progesterone ratio (days 19 to 21), with eight animals during the luteal phase, the period of lowest uterine blood flow and estrogen: progesterone ratio (day 13). The first day of behavioral estrus was designated as day 0. Uterine arteries from each pig were evaluated for (1) in vitro contractility to nerve stimulation, (2) alpha 1-adrenergic receptor binding with use of the specific ligand 3H-WB-4101, and (3) concentrations of norepinephrine with use of a radioenzymatic assay. Uterine arterial contractility to nerve stimulation was greater (p less than 0.01) for pigs in the luteal phase than for those in the follicular phase (216 +/- 36 versus 56 +/- 26 mm Hg). Furthermore, uterine arteries from luteal phase pigs had greater (p less than 0.05) alpha 1-receptor binding (47 +/- 6 versus 35 +/- 5 fmol/mg of protein) than those from follicular phase pigs. Uterine arterial concentrations of norepinephrine were similar for follicular phase and luteal phase pigs. These results suggest that ovarian steroids alter the function of uterine periarterial sympathetic nerves through changes in alpha 1-adrenergic receptor numbers, which may contribute to the marked changes in uterine blood flow observed during the porcine estrous cycle.

Quantitation of alpha 1-adrenergic receptors in porcine uterine and mesenteric arteries.

The activation of vascular alpha-adrenergic receptors may be involved in the control of uterine blood flow. A radioligand binding assay with the use of the alpha 1-adrenergic antagonist 3H-WB-4101 was established to characterize the alpha-adrenergic receptors in uterine and mesenteric arterial membranes obtained from nonpregnant pigs. Specific binding of 3H-WB-4101 was rapid, saturable, and exhibited the alpha-adrenergic agonist potency order of (-)-epinephrine inhibition constant [Ki] = 0.6 mumol/L greater than (-)-norepinephrine (Ki = 1.5 mumol/L) much greater than (-)-isoproterenol (Ki = 120 mumol/L). The alpha-adrenergic antagonist phentolamine (Ki = 6.0 nmol/L) was 200 times more potent than the beta-adrenergic antagonist (+/-)-propranolol (Ki = 1,200 nmol/L); the alpha 1-selective antagonist prazosin (Ki = 1.2 nmol/L) was 130 times more potent than the alpha 2-selective antagonist yohimbine (Ki = 160 nmol/L). Scatchard analysis, as well as iterative curve-fitting analysis, demonstrated that 3H-WB-4101 binding by arterial membranes was to a single class of binding sites. Uterine arteries exhibited greater maximal binding capacity (BMax) than that of mesenteric arteries (47.5 +/- 3.2 versus 30.9 +/- 3.6 fmol per milligram of protein, p less than 0.01), but the uterine artery dissociation constant (Kd) was higher, thus indicating a lower affinity, when compared with mesenteric artery (0.43 +/- 0.04 versus 0.33 +/- 0.04 nmol/L, p less than 0.05).

Effect of parasympathetic decentralization and paracervical ganglion excision on reproductive function in the rat.

The uterine paracervical ganglion (Frankenhauser's ganglion) contains the terminal ganglion cells of the sacral parasympathetic system and, in some species, the short adrenergic system. Histochemical studies also show numerous chromaffin cells with morphologic attributes of interneurons. The present study explores the function of cell types present in this ganglion, by seeking changes in reproductive function following either parasympathetic decentralization (transection of the cauda equina) or excision of the ganglion itself. Two well-known reproductive phenomena were observed after each surgical intervention, namely, induction of uterine hyperemia by estrogen administration, and maintenance of normal vaginal cycles. Estrogen-induced uterine hyperemia was not affected by parasympathetic decentralization or ganglion excision. Therefore, nerves originating in, or passing through this structure may be eliminated as components of the vascular control mechanism. In contrast, compared to sham-operated controls, ganglionectomy caused a significant reduction in the proportion of animals exhibiting normal vaginal cycles postoperatively (P less than 0.05). Cycle distribution was more evident in animals ganglionectomized on metestrus (P less than 0.01) and proestrus (P less than 0.05) than in animals ganglionectomized on diestrus or estrus. Since parasympathetic decentralization did not produce cycle disruption similar to ganglionectomy, one may conclude that the cycle-modulating effect does not involve preganglionic fibers of the sacral parasympathetic nerves.

Electron microscopic and histochemical characterization of intra-arterial cushions of the rat and porcine uterine vascular bed.

Scanning and transmission electron microscopic studies, together with histochemical investigations, were conducted on rat and porcine intra-arterial cushions from the uterine vascular bed. In the rat, the fine structure of these cushions closely resembled that previously described in the rat kidney. The cushions were composed of modified smooth muscle, circularly disposed in an incomplete, raised band surrounding the entrance to arterial branches. These muscle cells projected as attenuated processes throughout the loosely organized, PAS-positive stroma, and established close contact with thin endothelial extensions projecting from the base of the surface endothelial cells. Scanning electron microscopic observations of furrows on the endothelial surface gave rise to the suggestion that such contacts might mediate muscular control of endothelial surface topology. In similar cushions from the pig uterine artery, the smooth muscle of the cushions was much more compactly organized, and was disposed radially, rather than circumferentially, within the cushion structure. The enzyme histochemical profile of porcine cushions did not differ appreciably from that of normal vascular smooth muscle and endothelium, suggesting the maintenance of a metabolic similarity with adjacent tissues. These studies clarify the fine-structural basis for recently reported contraction and relaxation of uterine artery cushions during ischemia and perfusion of the rat uterine vascular bed, and thus, for their functional role in the regulation of uterine vascular flow.

Local and systemic effects of intrauterine estradiol-17 beta on luteal function of nonpregnant sows.

This study was conducted to determine if intrauterine injections of estradiol-17 beta (E2 beta) could maintain luteal function in nonpregnant sows. Eight sows were assigned to surgery on d 8 or 9 of the estrous cycle (first day of estrus = d 0). At surgery, cannulas were inserted bilaterally into the uterine artery (UA) and common utero-ovarian vein (UOV), as well as into the lumen of each isolated uterine horn. Electromagnetic blood flow transducers were placed around the middle uterine artery supplying each horn of four sows. After surgery, sows were assigned randomly to receive either intrauterine injections of vehicle (.9% NaCl) into both uterine horns (control sows) or E2 beta into one uterine horn (375 ng/injection) and vehicle into the other (treated sows) every 6 h from 1200 h on d 11 to 1200 h on d 15. Uterine blood flow (UBF) was quantified, and blood was sampled from the UA and UOV, periodically, from d 11 to 18. On d 18, sows were ovariectomized and corpora lutea (CL) were weighed. Blood plasma was subsequently analyzed for progesterone (P4) and prostaglandin F (PGF) by radioimmunoassay. Control sows had smaller (P less than .05) CL than treated sows on d 18 (3,046 +/- 614 vs 4,451 +/- 324 mg). Progesterone concentrations in UOV blood of treated sows tended to increase from d 11 (469 +/- 110 ng/ml) to 18 (626 +/- 209 ng/ml) while P4 in UOV blood of control sows decreased markedly (P less than .01) from d 11 (579 +/- 79 ng/ml) to 18 (14 +/- 5 ng/ml). In addition, UOV P4 concentrations on the E2-beta-injected side of treated sows were higher (P less than .05) than those on the vehicle-injected side from d 14 to 18. The UBF of two treated sows increased eightfold to 10-fold within 12 h of the first E2 beta injection and remained elevated through d 17, while UBF of two control sows remained constant. Prostaglandin F concentrations in UOV blood of treated sows were lower (P less than .05) than in UOV blood of control sows on d 14 and 15. There was no effect of side of E2 beta injection on PGF concentrations, which were similar in UOV blood draining both uterine horns of treated sows. Thus, the local effect of E2 beta in stimulating P4 secretion by the ipsilateral ovary is not due to reduced PGF concentrations in UOV blood draining the E2 beta-injected horn.

Effect of prostacyclin inhibition by tranylcypromine on uterine 6-keto-pgf 1 alpha levels during estrogen hyperemia in rats.

A role for prostacyclin (PGI2) as a mediator of estrogen-induced increases in uterine blood volume (UBV) was investigated by measuring uterine tissue levels of 6-keto-prostaglandin F1 alpha (6-keto-PGF 1 alpha), and testing estrogen responses in rats pretreated with the PGI2 synthesis inhibitor, tranylcypromine (TCP). Uterine 6-keto-PGF1 alpha content was determined by radioimmunoassay of tissue extracts purified through the use of high-performance liquid chromatography (HPLC). Estrogen treatment of castrate rats resulted in a significant increase of uterine 6-keto-PGF 1 alpha was compared to saline treated controls (9.3 ng/uterine horn vs 6.7 ng/uterine horn, p=0.01). Pretreatment with TCP (20 mg/kg) markedly reduced the uterine content of 6-keto-PGF 1 alpha (2.5 ng/uterine horn). The typical 50% increase in UBV observed after estrogen was unaffected by tranylcypromine pretreatment. It was concluded that the increased PGI2 synthesis, as indicated by elevated levels of 6-keto-PGF1 alpha, may function as an amplifying mechanism for the uterine vasodilation-induced by estrogen in castrate rats, but that production of this prostanoid is not essential for the estrogen response.

Plasma prostaglandin levels in radiation-induced enteritis.

Increased prostaglandin synthesis has been implicated as a causative factor in the production of radiation induced enteritis. Seventeen patients selected to begin pelvic irradiation for treatment of gynecological cancer had plasma Prostaglandin E, Prostaglandin F, and 13, 14 dihydro 15 keto PGF2 alpha metabolite determined by radioimmunoassay, prior to initiation of radiotherapy, at weekly intervals during treatment and at six weeks following completion of radiotherapy. A total of 362 prostaglandin determinations were performed. Thirteen patients (76%) developed significant diarrhea consisting of three or more watery bowel movements per day. Nine patients (53%) had intermittent colicky pain and six patients (35%) had nausea and vomiting during treatment. Statistical evaluation revealed no significant elevation of plasma prostaglandins during radiotherapy.

Effect of selective midbrain and diencephalic 5,7-dihydroxytryptamine lesions on serotonin content in individual preopticohypothalamic nuclei and on serum luteinizing hormone level.

Injection of the serotonin neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), into the midbrain dorsal (DR) or median (MR) raphe nucleus of castrated and normal male rats was followed by measurement of serum luteinizing hormone (LH) level and the 5-hydroxytryptamine (5-HT) content of several hypothalamic and amygdaloid nuclei. Only the DR lesions lead to a significant decrease (42%) in serum LH level in normal rats. The elevated LH level in castrated animals was not affected by either lesion. The DR lesions were followed by 5-HT reductions only in the medial preoptic area, the arcuate and ventromedial hypothalamic nuclei, and in the basal and central amygdaloid nuclei. In contrast, the 5-HT reductions produced by MR lesions were much more widespread, being found in all nuclei assayed with the exception of the dorso- and ventromedial hypothalamic nuclei. In a second experiment, degeneration of serotonergic terminals in the ventromedial region of the hypothalamus following intradiencephalic injections of 5,7-DHT led to a significant decrease in serum LH level and a 5-HT reduction in the arcuate, ventromedial and dorsomedial hypothalamic nuclei. 5,7-DHT injections into the medial preoptic area and the anterior hypothalamic area did not affect serum LH level. These results suggest that a serotonergic pathway originating in the midbrain dorsal raphe nucleus and innervating the mediobasal hypothalamus has a stimulatory influence on LH secretion.

Uterine catecholamines and prostaglandins during the estrous cycle of the rat.

The uterine contents of norepinephrine, epinephrine, dopamine, and prostaglandins F and E were determined on each day of the rat estrous cycle. Catecholamines were determined by high performance liquid chromatography with electrochemical detection (HPLC-EC) as well as by a radioenzymatic technique; prostaglandins were quantitated by RIA. The norepinephrine and dopamine values obtained by the radioenzymatic assay were approximately 1.5 times as high as the values obtained by HPLC-EC (norepinephrine, 285 vs. 188 ng/g; dopamine, 11.0 vs. 7.5 ng/g). Despite this difference in levels, both analytic techniques showed a decline in uterine norepinephrine from diestrus to estrus, followed by a significant (P less than 0.01) increase in norepinephrine on the day of metestrus. A cyclic pattern was also revealed for uterine dopamine concentration. There was a decline in dopamine concentration from diestrus to proestrus (radioenzymatic, P less than 0.01), followed by a return to high levels at metestrus (HPLC-EC). Epinephrine levels were low (undetectable by radioenzymatic assay; 24 ng/g by HPLC-EC) and showed no variation during the estrous cycle. Prostaglandin F was uniformly higher than prostaglandin E (10 vs. 2.5 ng/uterus). Significant increases in the uterine contents of both prostaglandins were shown on the day of proestrus.

Dysmenorrhea: treatment with an antiprostaglandin.

Painful menses, one of the most frequent gynecologic complaints, is incapacitating for many women. It has recently been proposed that increased endometrial prostaglandin production and prostaglandin-induced myometrial contractility may be responsible for dysmenorrhea. In this prospective, double-blind, 3-way, crossover study, relief of pain by an antiprostaglanding drug, ibuprofen (400 mg), was compared with propoxyphene (64 mg) and placebo in 22 women with severe primary dysmenorrhea. Ibuprofen was significantly more effective in 18 patients when compared to the other 2 treatment regimens (P less than 0.001), while propoxyphene was superior to placebo in 13 patients (P less than 0.05). Prostaglandin E and F synthesis rates in endometrial biopsy specimens taken on the second day of treatment in 2 patients during each treatment cycle were lowest during ibuprofen in one case but showed no definite pattern in the second.