Robert A. Corradino

Embryonic chick intestine in organ culture: stimulation of calcium transport by exogenous vitamin D-induced calcium-binding protein.

Abstract Vitamin D 3 or a potent metabolite, 1,25-dihydroxycholecalciferol, induces calcium-binding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.

Acute effects of prostaglandin F2α 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)

A comparison of the effects of cyclooxygenase prostanoids on progresterone 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.

Embryonic chick intestine in organ culture: Hydrocortisone and vitamin D-mediated processes

Abstract The effects of the glucocorticoid, hydrocortisone (HC), on vitamin D-mediated responses were examined in the organ-cultured, embryonic chick duodenum. In this system tissue responses to vitamin D-steroids in the culture medium include increased cAMP concentration, de novo synthesis of a specific calcium-binding protein (CaBP), enhanced uptake and transmucosal transport of calcium, and increased alkaline phosphatase activity. HC at levels ≥ 27.5 n m increased vitamin D-induced CaBP concentration: This apparently represents the first report of an interaction of HC with another steroid, vitamin D, in the regulation of the concentration of a specific protein. High levels of HC (≥27.5 μ m ) in the culture medium reduced duodenal calcium uptake and transmucosal transport regardless of the presence of vitamin D. However, at lower concentrations of HC (≤2.75 μ m ), only vitamin D-independent calcium uptake (basal calcium uptake) was reduced. Actinomycin D had no effect on HC reduction of basal calcium uptake suggesting that new protein synthesis is not involved in this action. In other experiments either HC or vitamin D stimulated phosphate and glucose uptake, and this uptake was potentiated by the presence of both steroids. HC also stimulated alanine uptake. Either HC or vitamin D increased both alkaline phosphatase activity (APA) and cAMP concentration, but together their activities were only additive. The data accumulated thus far indicate that HC directly influences calcium (and other nutrient) uptake by the duodenum and increases the concentration of the vitamin D-induced CaBP. Other vitamin D-mediated responses (APA and cAMP) were influenced by HC but there was no readily discernible relationship to nutrient uptake.

Cestrumdiurnum: A domestic plant with 1,25-dihydroxycholecalciferol-like activity

Summary The biological activity of Cestrum diurnum , in connection with the occurrence of calcinosis in grazing animals in Florida, was investigated. The inclusion of the plant material in a high strontium diet fed to chicks overcame the inhibitory effect of this regime on calcium (47Ca) absorption, the synthesis of the intestinal vitamin D-dependent calcium-binding protein, and net formation of intestinal cyclic adenosine-3′:5′-monophosphate. Since the high strontium diet blocks the conversion of 25-hydroxycholecalciferol to 1α,25-dihydroxycholecalciferol, this provides evidence of a substance in Cestrum diurnum with 1α,25-dihyroxycholcealciferol-like activity.

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 mac-roscopically 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.

Calcium-binding protein of intestine: Induction by biologically significant cholecalciferol-like steroids in vitro☆

Abstract Vitamin D (cholecalciferol-like) steroids added to the culture medium induce a specific calcium-binding protein (CaBP) ‡ in embryonic chick duodenum maintained in organ culture. Responses of the isolated duodenum faithfully mimic several other cholecalciferol-dependent responses observable in vivo attesting to their physiological significance. Therefore, this system provides a relevant bioassay, i.e. a physiological response (CaBP-induction) in a principal target organ, for the study of structure-activity relationships of biologically-significant cholecalciferol-like steroids. The several cholecalciferol-like steroids normally circulating in the intact animal, including cholecalciferol itself, 25-hydroxycholecalciferol, 24R,25-dihydroxycholecalciferol and 1α,25-dihydroxycholecalciferol, induced CaBP biosynthesis in vitro at or below physiological concentrations. This clearly demonstrates the relative rather than absolute specificity of the intestine for cholecalciferol-like steroids in the induction of CaBP. Maximal activity was observed with either 1α-hydroxycholecalciferol (not a native steroid), 1α,25-dihydroxycholecalciferol or 1α,24R,25-trihydroxycholecalciferol indicating a requirement for the 1α-hydroxyl function but suggesting the non-essentiality of the 25-hydroxyl function. The cis triene structure and an intact side-chain were also found to be essential to optimal activity. Preliminary studies with impure analogs suggest an absolute requirement for the 3β-hydroxyl group. The minimal structure inducing CaBP among naturally occurring steroids is cholecalciferol itself.

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.

Induction of calcium-binding protein in organ-cultured chick intestine by fluoro analogs of vitamin D3

Abstract Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D3 (D3) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D3) or in both the 1α and 25 positions (1α,25-F2-D3) were markedly more potent than vitamin D3 itself although 1α,25-F2-D3 was only 1 7 th as potent as 1α-F-D3. The 25-fluoro analog (25-F-D3) was a very weak inducer; only 1 45 th as potent as vitamin D3. The 25-fluoro analog of 1α-hydroxyvitamin D3 (1α-OH-25-F-D3) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24R-OH-25-F-D3 was about 15 times more potent than 24R,25-(OH)2-D3. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F2-25-OH-D3 and 24,24-F2-1α,25-(OH)2-D3 were about four times as potent as their nonfluorinated counterparts. The 24,24-F2-1α,25-(OH)2-D3 is, therefore, the most potent vitamin D3 analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D3 metabolite, 1α,25-(OH)2-D3.

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.