Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.     

Immunomodulatory actions of 1,25-Dihydroxyvitamin D3

The sterol, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), has immunosuppressive activity. The hormone inhibits the production of lymphokines (IL-2, IFN-γ) and monocyte-derived cytokine (IL-12) leading to inhibition of helper T cell subset type 1 (Th₁). When given in vivo, the hormone prevents the development of spontaneous and induced models of autoimmunity. Analogs of 1,25(OH)₂D₃, with reduced hypercalcemic effects, display an enhanced activity in autoimmunity compared to the sterol and prolong graft survival in experimental transplantation. This paper reviews our understanding of the cellular actions of the hormone and the therapeutic application of 1,25(OH)₂D₃ and analogs in autoimmunity and transplantation.     

Direct modulation of 25-hydroxyvitamin D3 hydroxylation in kidney tubules by 1,25-dihydroxyvitamin D3

Kidney tubules obtained from chicks fed a high-calcium low-phosphorus diet retained 25-hydroxyvitamin D₃-1-hydroxylase activity after a 10 h incubation in serum-free minimum essential medium. Inclusion of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in the medium prompted a suppression of 25-hydroxyvitamin D₃-1-hydroxylase and the induction of 25-hydroxyvitamin D₃-24-hydroxylase activities. The enzyme switch-over response could be prompted by 1.6 × 10^?7 M 1,25-dihydroxyvitamin D₃ and occurred within 6 h following treatment. Medium calcium appeared to augment the metabolite's switch-over action.     

Correlation between cyclic AMP levels and calcium efflux in isolated renal cortical tubules.

Isolated renal cortical tubules from male hamsters were utilized to examine the possible relationship between cyclic AMP (cAMP) and efflux of calcium. Both parathyroid hormone (PTH) and prostaglandin E1 (PGE1) produced dose-related increases in cAMP levels and calcium efflux from isolated tubules. Maximal concentrations of both hormones resulted in changes in cAMP which were 6 fold greater and changes in calcium efflux which were 2 fold greater with PGE1 than with PTH. Effects of sub-maximal amounts of either hormone on both cAMP and calcium efflux were potentiated to tubule incubations resulted in increases in tissue-associated cAMP over the same degree by inclusion of methyl-isobutylxanthine (MIX). Addition of either exogenous cAMP or dibutyryl cAMP (db-cAMP) produced dose-related increases in calcium efflux which occurred more rapidly with db-cAMP than with cAMP. Increasing amounts of cAMP added to the same concentration range resulting in increases in calcium efflux. Addition of 2', 3' cyclic AMP, 5'AMP or db-cyclic GMP had no significant effect on calcium efflux while 3', 5' cyclic CMP significantly reduced this response. The results indicate that cAMP increases efflux of calcium from renal tubules and may play a central role in hormone-dependent transport of this ion.     

Formation of 1,24-24-trihydroxyvitamin D3 from 1,25-dihydroxyvitamin D3

Incubation of [26,27-³H₂]-25-hydroxyvitamin D₃ with kidney homogenates from rats fed a high (3%) calcium vitamin D-supplemented diet results in the production of a more polar metabolite which cochromatographs with 1,24,25-trihydroxyvitamin D₃. On the other hand, incubation with kidney homogenates from vitamin D-deficient or calcium-deficient rats did not produce the polar metabolite. Mitochondria but not microsomes carry out the reaction and evidence has been produced to demonstrate that the 1,24,25-trihydroxyvitamin D₃ can be produced in vivo from either 1,25-dihydroxyvitamin D₃ as previously reported.     

A comparison of the metabolism of 25-hydroxyvitamine D3 by chick renal tubules, homegenates and mitochondria

The metabolism of 25-hydroxycholecalciferol by isolated chick renal tubules has been investigated by the use of a new chromatographic method employing silica-treated paper as the supporting phase. This method has been found to be superior to Sephadex LH20 column chromatography in resolving the metabolites of 25-hydroxycholecalciferol, particularly in separating 1,25-dyhydroxycholecalciferol from other metabolites. With this method several heretofore undescribed metabolites of 25-hydroxycholecalciferol have been recognized and partially characterized.     

Effects of parathyroid hormone on cyclic AMP, cyclic GMP, and efflux of calcium in isolated renal tubules.

The effects of parathyroid hormone (PTH) on concentrations of cyclic AMP and cyclic GMP were investigated in isolated renal cortical tubules from hamsters. Efflux of 45Ca from tubules was compared to temporal changes in both cyclic nucleotide concentrations. A rapid increase in cyclic AMP occurred following addition of PTH which was maximal by 1 min but decreased over the next 4 min period. Cyclic GMP concentrations were not significantly altered at 1 min but increased between 1 and 5 min from basal levels. Concentrations of both nucleotides remained significantly elevated from basal levels between 5 and 15 min following PTH. Efflux of 45Ca was increased by PTH with time-course changes closely paralleling changes in cyclic GMP concentrations. Changes in both cyclic AMP and cyclic GMP were related to PTH concentrations of the incubation media and were increased by addition of theophylline. Increasing the calcium concentration from 1 to 3 mM did not significantly alter the effect of PTH on cyclic AMP, however, cyclic GMP concentrations were further increased.     

Neuroeffector actions of prostaglandin D2 on isolated dog mesenteric arteries

Treatment with prostaglandin (PG) D₂ in concentrations (10⁻⁸ to 10⁻⁷ M) insufficient to alter the basal tone potentiated the contractile response of helical strips of dog mesenteric arteries to transmural electrical stimulation but did not influence the response to norepinephrine. The potentiating effect of PGD₂ was not prevented by treatment with diphloretin phosphate, a PG antagonist, whereas contractions of dog cerebral arteries induced by PGD₂ were suppressed. The ³H-overflow evoked by transmural stimulation in superfused mesenteric arterial strips previously soaked in ³H-norepinephrine containing media was significantly increased by PGD₂. It is concluded that PGD₂ increases the stimulation-evoked release of norepinephrine from adrenergic nerves innervating the arterial wall. PGD₂ appears to act differently on receptive sites responsible for increasing the release of norepinephrine and for producing arterial contraction.     

Comparative behavioral effects of dibutyryl cyclic AMP and prostaglandin E1 in mice

Three behavioral tests, spontaneous locomotor activity (SLMA), exploratory behavior (EB) and rotarod performance (RP), a measure of neuromuscular coordination, were used to study the interaction of PGE₁ (1 mg/kg i.p., 10 min. pretreatment) with DBcAMP (25 mg/kg i.p., 25 min. pretreatment) in mice. A dose-response relationship of PGE₁ (0.01–5.0 mg/kg) to SLMA was determined, with a significant decrease in SLMA produced by a dose of 0.1 mg/kg. Decreases in SLMA were produced by PGE₁ (79%), DBcAMP (41%) and DBcAMP-PGE₁ combination (71%). Similar decreases in EB were observed. Although no significant difference between controls and DBcAMP was observed in RP, 52% of mice tested were RP failures following PGE₁ and a 100% failure rate was induced by the combination. Mice were treated with a second injection of DBcAMP or PGE₁ or the combination 24 hr following the first injection. Behavioral activity of these mice was observed 25 min (DBcAMP) or 10 min (PGE₁) after the second dose was administered. A second injection of DBcAMP failed to decrease SLMA and EB from controls; moreover, SLMA began to return towards control levels as early as 2 hr between injections. The second injection of PGE₁ or DBcAMP+PGE₁ produced the same behavior as that produced by the first injection. On the basis of these results, the relationship of cyclic nucleotides and PGs to behavioral activity is discussed.     

Immunohistochemical Analysis of 1,25-dihydroxyvitamin D3 Receptor in Cervical Carcinoma

The immunohistochemical localization and expression of 1,25-dihydroxyvitamin D₃ receptors (VDR) has been investigated in normal human cervical tissue (n = 15) and in cervical carcinomas (n = 23). VDR immunoreactivity (monoclonal antibody 9A7r354;) was compared with the staining patterns of transglutaminase K, cytokeratin 10 and Ki-67 in these tumours. Moderate to strong nuclear immunoreactivity for VDR was detected in almost all cervical carcinomas analysed. VDR staining was homogeneous, with no visual differences between individual tumour cells. Some 60% of normal cervical tissues revealed weak immunoreactivity for VDR. In normal cervical tissue, nuclear VDR staining was confined to the lower cervical layers, predominantly to the basal cell layer. Both the intensity of VDR immunostaining and the number of VDR-positive cells were up-regulated in cervical carcinomas compared with normal cervical tissue. No visual correlation wa s found for the coexpression of VDR with markers of proliferation and differentiation. Our findings indicate that: (1) cervical tissue may be a new target organ for therapeutically applied vitamin D analogues; (2) VDR is up-regulated at the protein level in cervical carcinomas compared with normal cervical tissue; (3) up-regulation of VDR in cervical carcinoma is induced not exclusively by alterations in epithelial differentiation or proliferation, but by different, unknown mechanisms; and (4) calcitriol and new vitamin D analogues exerting fewer calcaemic side-effects may be promising new drugs for the treatment or chemoprevention of metastasizing cervical carcinomas as well as of cervical precancerous lesions.     

The actions of leukotrienes C4 and D4 in the porcine renal vascular bed

The kidney of anaesthetised pigs was perfused $\text{in situ}$ with carotid arterial blood. Renal blood flow and perfusion pressure were recorded. Close intra-arterial injection of leukotriene (LT) C₄, D₄ or noradrenaline (NA) caused a dose-related increase in vascular resistance. Both LTs were more active than NA by one to two orders of magnitude. Systemically-administered indomethacin potentiated the effect of all three agonists. Incubation of renal artery tissue with calcium ionophore A23187 in the presence of indomethacin resulted in the generation of LT-like material which, when assayed on guinea-pig ileum, was indistinguishable from LTD₄. The results show that pig renal vessels produce LT-like material and suggest that the potent vasoconstriction induced by exogenous NA and LTs is modulated $\text{in vivo}$ by a vasodilator cyclo-oxygenase product.     

Effects of probenecid on transport and metabolism of cyclic AMP by isolated rabbit renal tubules

The effects of probenecid on the transport and metabolism of cyclic [14C]-AMP were studied in isolated rabbit kidney cortex tubules. Incubation in a medium with 10-400 microM probenecid for 30 min caused a 30-70% decrease in the tubular uptake of labeled material from a medium containing 0.1 mM cyclic [14C]AMP. The radioactivity in the tubules, after 30 min incubation, with or without probenecid, was mostly in the form of inosine and hypoxanthine. The disappearance of external cyclic [14C]AMP was retarded by probenecid and the concentration ratio of cyclic AMP to inosine + hypoxanthine was increased. Cyclic AMP phosphodiesterase activities, from both the soluble and particulate fractions of the kidney, were inhibited by probenecid. These findings indicate that the changes caused by probenecid on the renal disposal of extracellular cyclic AMP can be accounted for by a decrease in the accumulation of the products of cyclic AMP metabolism secondary to inhibition of extracellular cyclic AMP phosphodiesterase activity.     

Intestinal 1,25-dihydroxyvitamin D3 binding protein: Specificity of binding

The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ are equally competitive and are the most active. Of the structural features of the compounds, the 1α-hydroxyl is most important followed by the 25-hydroxyl and the 3β-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D₃ was much more effective than vitamin D₃ but less effective than 25-hydroxyvitamin D₃. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D₃ binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D₃ and the cytosol receptor.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Stimulation of cyclic AMP production in chick renal tissue by cadmium and manganese

The effects of cadmium on production of cyclic AMP by partially purified chick renal plasma membrane preparations and binding of 125I-parathyrin to the membranes have been investigated. At certain concentrations Cd2+ ions (and Mn2+ ions) markedly stimulated the production of cyclic AMP by the tissue. It was found that concentrations of Cd2+ roughly in the same range were also capable of stimulating binding of 125I-parathyrin to the membrane preparations.     

Metabolism of cyclic AMP in isolated renal tubules: Effects of prostaglandins and parathyroid hormone

Concentrations of cyclic AMP (cAMP) were increased in isolated renal cortical tubules from hamsters by both parathyroid hormone (PTH) and prostaglandin E₁ (PGE₁) with maximal effects of PGE₁ being 6–8 fold greater than those of PTH during a 10 min period. However, cAMP concentrations in cells treated with 1-methyl-3-isobutylxanthine (MIX) were increased with maximal concentrations of either hormone to the same degree. Similar effects of both hormones were observed on adenylate cyclase activity in renal homogenates. Simultaneous addition of hormones produced changes in both cAMP concentrations in intact tubules as well as adenylate cyclase activity of homogenates which were not completely additive. Degradation of cAMP, estimated in intact tubules as the difference in cAMP levels in the presence and absence of MIX, was increased by both hormones, however, changes were 2–3 fold greater in tubules exposed to PTH than to PGE₁. Neither hormone directly altered cAMP phosphodiesterase (PDE) activity in either 30,000 x g supernatant or pellets from renal cortical homogenates.The results suggest that both hormones increase the production of cAMP in renal cortical tubules and may share a common target cell type in this response. Degradation of cAMP, however, is differentially effected by the two hormones, probably reflecting differences exerted on intracellular mechanisms regulating the enzymatic hydrolysis of cAMP.     

The clearance of human fibrinogen fragments D1, D2, D3 and fibrin fragment D1 dimer in mice

The clearance of human fibrinogen fragments D₁, D₂, D₃ and fibrin fragment D₁ dimer were studied in the mouse model. Clearance of these fragments is a complex process involving clearance from blood into three other compartments. The overall clearance of fragment D₁ and its dimer were essentially identical. Fragments D₂ and D₃ cleared at a progressively slower rate. Competition studies were performed between ¹²⁵I-labeled fragment D₁ and large molar excesses of unlabelled human fragments D₁, D₂, D₃, D₁ dimer, fragment E, fibrinogen, macroalbumin, mannan and asialooroscomucoid. Of these ligands only the fragment D variants competed for the clearance of ¹²⁵I-labeled fragment D₁. Cross-competition was observed when ¹²⁵I-labeled fragment D₁ dimer was cleared in the presence of large molar excesses of fragment D₁. Autopsies demonstrated that injected fragments D₁, D₂, D₃ and D₁ dimer cleared primarily in liver and kidneys. In some clearance studies, livers were perfused with tissue culture fluid, subjected to light microscopic autoradiography, and silver grain counts performed to localize cleared fragment D₁. These experiments indicated that 80% of the liver uptake was in hepatocytes. However, when silver grain counts were normalized for the number of parenchymal and nonparenchymal cells, the distribution of silver grains was essentially identical (1.8 and 1.6 grains per cell, respectively). It is concluded that fragments D₁, D₂, D₃ and D₁ dimer are recognized by a similar clearance pathway. Since neither fibrinogen nor fragment E competed for the clearance of fragment D₁, it is suggested that determinants present in the fragment D domain become exposed after plasmin attack on fibrinogen and are responsible for clearance.     

Cardiovascular effects of prostaglandin D3 and D2 in anesthetized dogs

Prostaglandin (PG) D₃ has been identified as an inhibitor of human platelet aggregation, but little is known of the hemodynamic activity of this material. In morphine pretreated, chloralose-urethan anesthetized dogs, bolus intravenous injections (1, 3.2 and 10 μg/kg) of PGD₃ and also PGD₂ were associated with marked, dose-related increases in pulmonary arterial pressure. Cardiac index and rate increased, while peripheral vascular resistance decreased in response to injections of PGD₃. A biphasic (depressor followed by a pressor phase) effect on systemic arterial pressure was observed after PGD₂, while PGD₃ was associated with dose-related depressor responses. Graded intravenous infusions (0.25, 0.50 and 1.0 μg/kg/min) of PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses. Quantitatively, PGD₃ infusions were associated with greater decreases in peripheral vascular resistance and greater increases in cardiac output, heart rate, and peak left ventricular dp/dt than were infusions of PGD₂. In contrast, PGD₃ was less potent than PGD₂ as a pulmonary pressor material. Systemic arterial pressure responses to infusions of the prostaglandins were variable. In these experiments, PGD₃ and PGD₂ were associated with qualitatively similar cardiovascular responses characterized by peripheral vasodilatation.