Growth factor receptors gene expression and Akt phosphorylation in benign human thyroid nodules are unaffected by chronic thyrotropin suppression.

Levothyroxine (L-T4)-based suppression of thyrotropin (TSH) secretion is widely used to prevent the growth of benign thyroid nodules, although the effectiveness of this approach has been demonstrated only in a subset of patients. In this study, we analyzed the in vivo effects of L-T4-mediated TSH suppression on elements of insulin/IGF-1-dependent growth-regulating pathways in tissues from patients with benign thyroid nodules. Nodular and non-nodular tissue specimens were collected from 63 patients undergoing thyroidectomy. 32 had received preoperative TSH suppressive therapy with TSH levels consistently below 0.5 mU/l (L-T4 group). TSH suppression had not been used in the other 31, and their TSH levels were normal (0.8-4 mU/l (control group). Quantitative RT-PCR was used to measure mRNA levels for TSH receptor, IGF1, IGF-1 receptor, insulin receptor, insulin receptor substrate 1 in nodular and non-nodular tissues from the 2 groups. Akt and phosphorylated Akt protein levels were detected by Western blot. Mean levels of mRNA for all genes tested were similar in the 2 groups, in both nodular and non-nodular tissues. The 2 groups were also similar in terms of phosphorylated Akt protein levels (measured by densitometric scan in 10 randomly selected nodules from each group). This is the first demonstration based on the study of human thyroid tissues that TSH suppression does not affect the expression of components of the insulin/IGF-1-dependent signaling pathways regulating thyrocyte growth. This may explain the lack of effectiveness of TSH-suppressive therapy in a substantial percentage of benign thyroid nodules.

[On the job training at construction sites of the hospital corporation of Careggi (Florence)]. / Tutoraggio formativo in itinere nei cantieri edili dell'Azienda Ospedaliera di Careggi (Firenze).

The "Careggi Safety" project has like objective to guarantee the safety execution of Careggi Hospital (Florence) restoration and development works, thanks to an in itinere training program into building sites and through tutor figure using. The project aim is to overcomes traditional indoor training limits, not effective in complex and dynamics reality like as building sites, constrained by contracts deadline and high labour turnover (subcontracts) inside carry out process. Solutions chose are: (a) a training projected in itinere, following site works evolution and safety and coordination plan, and through a constant agreement between customer and operative enterprises; (b) a building site's tutor, standing beside workers during realization phases, contributing to form on respective safety carry out job. In to "Careggi Safety" project training has been chose as preliminary and obligatory condition for labours admission and control into the building site.

[A case of gastric lipoma. Diagnostic imaging]. / Presentazione di un caso di lipoma gastrico. Imaging diagnostico.

A case of gastric lipoma which manifested with an episode of acute gastrointestinal hemorrhage is reported. Preoperative diagnosis was based on the US, TC, and MRI findings, as the results of gastrointestinal endoscopy were inconclusive. The role of current imaging methods, and particularly of MRI, is discussed.

[Characterization of non-secreting adrenal nodules (incidentalomas): role of multiphasic spiral computerized tomography]. / Caratterizzazione dei noduli surrenalici non secernenti (incidentalomi): ruolo della Tomografia Computerizzata spirale multifasica.

PURPOSE: To evaluate the diagnostic yield of multiphasic helical CT in the characterization of single non functioning adrenal nodules (incidentalomas) less than 50 mm in diameter. Emphasis was given to the possible replacement of unenhanced with delayed scans in cancer patients undergoing staging procedures. MATERIAL AND METHODS: Sixty patients with single adrenal nodules (30 of them neoplastic and 30 non-neoplastic) were examined with thin unenhanced scans (5 mm), early scans after administration of a contrast agent (120 mL at 2.5 mL/s with 60 s delay) and late scans (30 min delay). RESULTS: On both unenhanced and late scans a threshold could be selected on the Hounsfield unit scale which guaranteed absolute specificity in the characterization of adenomas (100% specificity) with very high sensitivity (93% at both scans): this threshold was 19 HU on unenhanced and of 41 HU on late scans. In contrast, at early delayed scanning the threshold which guaranteed 100% specificity was associated with negligible sensitivity (30%). The evaluation of lesion size had no diagnostic value, since the mean diameter of both benign and malignant nodules was of 25 mm. CONCLUSIONS: Late scans have diagnostic yield comparable to unenhanced scans: at the selected delay (30 min), benign lesions nearly always have lower attenuation values than malignant nodules and can be diagnosed with confidence when they exhibit mean attenuation values lower than 41.

[Gallbladder carcinoma. Our experience]. / Carcinoma della colecisti. Nostra esperienza.

The authors report their experience of 18 patients with primary cancer of the gallbladder. On 10 patients at stage IV, 9 had a preoperative diagnosis, while at stage 0-1 and 2 the diagnosis was intraoperative or histologic. Every patient had a cholelithiasis at the same time. The authors discuss prophylactic cholecystectomy, even without specific symptoms, and emphasize the need for a better morphological and radiomorphological classification. In the light of the new microinvasive surgical techniques, they briefly discuss laparoscopic cholecystectomy and histologic diagnosis of carcinoma of the gallbladder.

Calbindin D28K regulation in precociously matured chick egg shell gland in vitro.

Egg shell calcification in the hen uterus (egg shell gland, ESG) depends primarily on intestinal absorption of dietary Ca2+ as well as ESG Ca2+ transport into the shell. Intestinal Ca2+ absorption is linked to vitamin D-induced calbindin D28K (D28K) concentration. The ESG also contains D28K, and Ca2+ transport into the shell appears to be linked to D28K gene expression, but until this report, there was no direct proof that ESG D28K was or was not vitamin D-dependent. To address this issue, highly developed ESG from estradiol (E2)-injected, severely vitamin D-depleted chicks were cultured in serum-free medium with excellent viability. Addition of the vitamin D-hormone, 1,25(OH)2 vitamin D3 (1,25), to the culture medium increased ESG D28K levels as much as 70%. E2 alone had no effect, but E2 plus 1,25 further increased ESG D28K levels up to 160%. By contrast, progesterone (P4) prevented the 1,25-stimulated increase in D28K, while having no effect on basal D28K level. Of considerable interest, thapsigargin (THAPS), which increases intracellular Ca2+ concentration ([Ca2+]i) in many cell types, stimulated D28K synthesis in a concentration-dependent manner in the complete absence of 1,25 and independent of the [Ca2+] of the medium. These results are the first direct evidence that ESG D28K is under direct control of 1,25 and that both gonadal steroid hormones, E2 and P4, may be coregulators. Further, the effects of THAPS suggest that [Ca2+]i itself may also regulate D28K. This new in vitro model clearly represents a unique opportunity to study the regulation of the ESG calcium transport mechanism under stringently defined conditions.

Tissue-specific regulation of shell gland calbindin D28K biosynthesis by estradiol in precociously matured, vitamin D-depleted chicks.

Provision of Ca2+ for egg shell calcification in the avian uterus [egg shell gland (ESG)] derives mostly from vitamin D-dependent intestinal Ca2+ absorption from the diet. Ca2+ absorption is strongly linked to the intestinal vitamin D-dependent calbindin D28K (D28K) concentration. The laying hen ESG also contains D28K, and again, Ca2+ transport into the shell appeared to be linked to the ESG D28K concentration. However, evidence is now presented that ESG D28K synthesis may be estradiol (E2) dependent and vitamin D independent under certain conditions. One-day-old female chicks fed a vitamin D-free diet for as long as 6 weeks and then repeatedly injected im with E2 for up to 3 more weeks developed frank rickets, but possessed precociously matured reproductive tracts. While the tiny presumptive ESGs of nonestrogenized vitamin D-depleted chicks were devoid of D28K, the highly developed ESG, including the isthmus, of estrogenized chicks contained D28K. The ESGs of nonestrogenized, vitamin D-replete chicks also exhibited no development or detectable D28K. Regardless of whether vitamin D depleted or replete, estrogenized chick ESG contained similar D28K and D28K mRNA concentrations. Immunohistochemical techniques showed that the endometrial cellular localization of both D28K and Ca(2+)-ATPase (Ca2+ pump) in estrogenized chicks was similar to that in mature laying hens. There was no trace of D28K, nor was there any stimulation of Ca2+ absorption, in duodenum of vitamin D-free, immature chicks regardless of E2 treatment. As expected, both D28K and D28K mRNA were present in vitamin D-replete chick duodenum. We conclude that in E2-treated chicks, ESG D28K gene expression may be vitamin D independent and E2 dependent. This is the first clear demonstration of hormone-dependent tissue-specific D28K gene expression in the chick.

Positive cotranscriptional regulation of intestinal calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 and glucocorticoids.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces expression of the gene encoding calbindin-D28K, a protein involved in intestinal Ca2+ transport. Glucocorticoids stimulate intestinal development and function, and presumed interaction with 1,25-(OH)2D3 has been intensively studied. Most studies involved administration of high doses of glucocorticoids in vivo, which inhibits intestinal Ca2+ transport by an unknown mechanism. However, it is now known from studies of the duodenal organ culture model that low concentrations of glucocorticoids enhance 1,25-(OH)2D3-dependent calbindin-D28K biosynthesis and Ca2+ transport. High concentrations mimic the action of administered glucocorticoids in vivo, suggesting that a distinct pharmacological or toxic mechanism causes inhibition of Ca2+ absorption. This report further shows that dexamethasone (DEX) rapidly enhanced calbindin-D28K gene expression, that is de novo calbindin-D28K mRNA biosynthesis. DEX also markedly reduced the actions of RNA and protein synthesis inhibitors on calbindin-D28K gene expression, although no evidence for an action of DEX or 1,25-(OH)2D3 at the translational level was obtained. Ca2+ transport activity was highly correlated with calbindin-D28K concentration regardless of treatment. Washout permitted complete reversal of inhibition, verifying the specificity of inhibitor activity. These results appear to show positive contranscriptional regulation of calbindin-D28K gene expression by 1,25-(OH)2D3 and glucocorticoids. The use of this model should continue to clarify the interactive roles of nuclear-acting hormones on the Ca2+ absorptive mechanism and on complex physiological and pathological processes in general.

Differential effects of calcium on progesterone production in small and large bovine luteal cells.

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.

The effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study.

Phenylbutazone, a non-steroidal anti-inflammatory drug known to produce intestinal erosions, was administered intravenously (13.46 mg/kg bodyweight) to 12 horses which were killed after 24, 48, 72 and 96 h. Eight untreated horses served as controls. Annular erosions in the duodenum and mucosal necrosis in the colon were seen after 48 h which progressed in severity. The erosions were characterised by sloughing of the surface epithelium, subepithelial cleft and bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries and microthrombosis. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Ultrastructurally, there was swelling of the endothelium of capillaries and small vessels, and of pericyte and smooth muscle cytoplasm in arterioles. In capillaries and post capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and oedema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury associated with the formation of duodenal and colonic erosions in horses. The duodenal and colonic mucosa were assayed at 48 and 96 h for prostacyclin and PGE2. There was no statistically significant difference between prostaglandin levels in the mucosa of control and treated horses. It was concluded that there was no correlation between mucosal prostaglandin levels and intestinal erosions after 48 h.

The effects of phenylbutazone on the morphology and prostaglandin concentrations of the pyloric mucosa of the equine stomach.

Phenylbutazone, a nonsteroidal anti-inflammatory drug known to produce gastric ulcers, was administered intravenously (13.46 mg/kg body weight) daily to 12 horses. Horses were euthanatized daily after 24, 48, 72, and 96 hours following the initial injection. Eight untreated horses served as controls. Small multifocal pyloric erosions were seen after 24 hours and then progressed in severity over time. The erosions were characterized by sloughing of the surface epithelium, subepithelial bleb formation, necrosis of the lamina propria, degeneration of the walls of subsurface capillaries, and microthrombosis of the capillaries of the pyloric mucosa. Large numbers of neutrophils with abundant fibrin and cellular debris were present at the erosion sites. Eroded pyloric mucosa and adjacent macroscopically intact mucosa were examined ultrastructurally. In both the macroscopically eroded mucosa and multifocally in the adjacent macroscopically uneroded mucosa, there was cellular swelling of the endothelium, pericytes, and smooth muscle cells of arterioles. In capillaries and post-capillary venules, the endothelium ranged from swollen to lysed and necrotic. Extensive extravasation of erythrocytes and edema were seen. These lesions were not seen in the control horses. Phenylbutazone produces a microvascular injury that is associated with the formation of pyloric erosions in horses. The pyloric mucosa of six horses was assayed for prostacyclin and prostaglandin E2 at 48 and 96 hours following the initial injection. There was no statistically significant difference between prostaglandin concentrations in the mucosa of control and treated horses. It was concluded that there was little correlation between pyloric mucosal prostaglandin concentrations and pyloric erosions after 48 hours.

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells.

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca2+ probe, Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca2+]i in both small and large luteal cells, having an effect at concentrations as low as 5 microM; and was maximally effective at 50 microM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca2+]i, with NDGA exerting a greater stimulation of [Ca2+]i than indomethacin. Prostaglandin F2 alpha (PGF2 alpha) as well as prostaglandin E2 (PGE2) increased [Ca2+]i in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF2 alpha and PGE2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.

Synthesis and biological activity of novel vitamin D analogues: 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 and 24,24-difluoro-1 alpha,25-dihydroxy-26,27-dimethylvitamin D3.

We synthesized 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 (16), and 24,24-difluoro-1 alpha, 25-dihydroxy-26,27-dimethylvitamin D3 (21), from 3 beta-hydroxy-22,23-dinorcholenic acid 3-acetate. Compound 16 was found to be a highly potent vitamin D analogue with bioactivity similar to that of 25-hydroxyvitamin D3 in vivo. Compound 16 was bound by vitamin D binding protein with an affinity slightly less than that of 25-hydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with approximately the same affinity as that of 25-hydroxyvitamin D3. In the organ-culture duodenum, 16 induced the synthesis of calcium binding protein with a potency approximately 1/20 that of 1,25-dihydroxyvitamin D3. Compound 21 was also noted to be a highly potent vitamin D analogue with bioactivity in vivo similar to that of 1,25-dihydroxyvitamin D3. It was bound to vitamin D binding protein with an affinity considerably less than that of 1,25-dihydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with an affinity slightly less than that of the native hormone. In the organ-culture duodenum, 21 was noted to be about 3 times more active than 1,25-dihydroxyvitamin D3 in the induction of calcium binding protein. The introduction of fluorines at C-24 and extension of the sterol side chain at C-26 and C-27 by methylene groups results in vitamin D analogues that have biological activity in vivo similar to those of the respective nonfluorinated natural sterols.

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a.

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.

Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells.

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.

Ascorbate interacts with sodium selenite to increase glutathione peroxidase activity in selenium-deficient chick duodena cultured in vitro.

Duodena from Selenium (Se)/vitamin E-depleted 19 d chick embryos were cultured in vitro for 0-30 h. The addition of sodium selenite to the culture medium was associated with increased selenium-dependent glutathione peroxidase (SeGSHpx) activity after 24 h of incubation. In the absence of Se or in the presence of sodium ascorbate supplementation alone, SeGSHpx activity showed a gradual decline over the same time period. When ascorbate was added, along with sodium selenite, SeGSHpx activity was increased earlier and to a greater extent than in the presence of Se alone. These observations show that ascorbate can influence the metabolism of sodium selenite, resulting in increased SeGSHpx activity.

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D3 and 1,25-dihydroxy-26,27-dimethylvitamin D3: highly potent novel analogs of vitamin D3.

We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 14 was also highly active in vivo. At doses of 1000-5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.

A comparison of the effects of cyclooxygenase prostanoids on progesterone production by small and large bovine luteal cells.

Highly purified preparations of small and large bovine luteal cells were utilized to examine the effects of prostaglandins F2 alpha (PGF2 alpha), E2 (PGE2) and I2 (PGI2) analog on progesterone production. Corpora lutea were obtained from Holstein heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. Progesterone accumulation was determined in 1 x 10(5) small and 5 x 10(3) large cells after 2 and 4 h incubations respectively. Progesterone synthesis was increased (p less than 0.05) in the small cells by the increasing levels of PGF2 alpha, PGE2, carba-PGI2 and LH. PGF2 alpha, but not PGE2 or carba-PGI2 increased (p less than 0.05) LH-stimulated progesterone production. There was no interaction of various combinations of prostaglandins on progesterone production in the small cells. In the large cells, PGF2 alpha had no effect on basal progesterone production. However, it inhibited LH-stimulated progesterone synthesis. In contrast, PGE2 and carba-PGI2 stimulated (p less than 0.05) basal progesterone production in the large cells. In the presence of LH, high levels of carba-PGI2 inhibited (p less than 0.05) progesterone synthesis. The PGE2 and PGI2-stimulated progesterone production in the large luteal cells was also inhibited in the presence of PGF2 alpha. These data suggest all of the prostaglandins used exert a luteotropic action in the small cells. In the large cells only PGE2 and carba-PGI2 are luteotropic, while PGF2 alpha exerts a luteolytic action. The effects of the prostaglandins in the small and large luteal cells suggest that their receptors are present in both cell types.

Acute effects of prostaglandin F2 alpha on inositol phospholipid hydrolysis in the large and small cells of the bovine corpus luteum.

The present studies were conducted to determine whether the large or small bovine luteal cell was the site for the stimulatory effect of prostaglandin F2 alpha (PGF) on phospholipase C-catalyzed inositol phospholipid hydrolysis. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and large luteal cells were isolated by flow cytometry using a Becton Dickson FACS 440 cell sorter. PGF provoked rapid (5-30 s) and sustained (up to 30 min) increases in the levels of inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively) in small luteal cells. IP3 was formed more rapidly than IP2 or IP following PGF treatment. The PGF-stimulated increase in IP3 was accompanied by a transient reduction in the levels of 3H-labeled phosphatidylinositol 4,5-bisphosphate. LiCl (10 mM) enhanced inositol phosphate accumulation in response to PGF. Maximal increases in inositol phosphate accumulation were observed with 1-10 microM PGF and half-maximal increases were observed with 60 nM PGF. PGF (1-10 microM) had no effect on cAMP levels but stimulated small increases in progesterone accumulation in 30 min incubations of small luteal cells. PGF also increased the accumulation of inositol phosphates in large luteal cells. The increases were apparent within 5 min of incubation (the earliest time examined) and further increases were observed in incubations lasting 30 min. PGF had no significant effect on cAMP or progesterone in 30 min incubations of large cells.(ABSTRACT TRUNCATED AT 250 WORDS)