Relationship between free and total 1,25-dihydroxyvitamin D in conditions of modified binding.

OBJECTIVE: A novel assay was employed to study the free 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations in various populations with different levels of 1,25(OH)2D and vitamin D binding protein (DBP). DESIGN: In 12 healthy women before and after 3 months of oral estrogen/progestagen treatment, 10 pregnant women, and 16 patients with a nephrotic syndrome and normal renal function, the concentrations of total and free 1,25(OH)2D, DBP and albumin were assessed. METHODS: The total concentration of 1,25(OH)2D in serum was assessed using a radioreceptor assay. The free fraction of 1,25(OH)2D was measured using symmetric dialysis. DBP was assessed using single radial immunodiffusion. Serum albumin concentrations were measured on an automated analyzer. RESULTS: In healthy women, the concentrations of total 1,25(OH)2D, free 1,25(OH)2D and DBP were 132+/-19 pmol/l, 92+/-30 fmol/l and 5.59+/-0.43 micromol/l. After 3 months of estrogen/progestagen treatment, total 1,25(OH)2D and DBP levels rose significantly to 175+/-51 pmol/l and 8.32+/-1.59 micromol/l (P< or =0.05 and P< or =0.001); the free 1,25(OH)2D remained unchanged (105+/-39 fmol/l; not significant). Pregnant women had significantly higher levels of total 1,25(OH)2D and DBP (239+/-68 pmol/l and 11.32+/-1.77 micromol/l; both P

Female sex hormone replacement therapy increases serum free 1,25-dihydroxyvitamin D3: a 1-year prospective study.

OBJECTIVE: Currently, hormone replacement therapy is applied successfully to reduce post-menopausal bone resorption. However, the exact mechanism by which oestrogen exerts its effect has not yet been fully elucidated. In order to determine whether changes in the biologically active 1,25-dihydroxyvitamin D3 may be of importance in this process, the concentrations of both total and free 1,25-dihydroxyvitamin D3 in serum were assessed. DESIGN: In 36 post-menopausal women the effect of hormone replacement therapy, with a combination of 17 beta-oestradiol and norethisterone acetate, on the serum levels of total and free 1,25-dihydroxyvitamin D3 was studied after 0, 3, 6 and 12 cycles. MEASUREMENTS: The total concentration of 1,25-dihydroxyvitamin D3 in serum was assessed using a radioreceptor assay after diethylether extraction of the samples followed by paper chromatography. The free fraction of 1,25-dihydroxyvitamin D3 was measured using symmetric dialysis. The free 1,25 dihydroxyvitamin D3 concentration was calculated by multiplying the total concentration by the free fraction. RESULTS: During therapy, mean serum total 1,25-dihydroxyvitamin D3 concentrations (+/- SD) were 106.4 pmol/l (+/- 27.5), 155.0 pmol/l (+/- 49.5), 176.7 pmol/l (+/- 70.0) and 161.1 pmol/l (+/- 55.3) at 0, 3, 6 and 12 cycles, respectively. Serum free 1,25-dihydroxyvitamin D3 concentrations were 68 fmol/l (+/- 22), 107 fmol/l (+/- 35), 120 fmol/l (+/- 43) and 108 fmol/l (+/- 37), respectively. Baseline values of both total and free 1,25-dihydroxyvitamin D3 were significantly lower than those during therapy at all time (P < or = 0.001). CONCLUSION: Both the serum total 1,25-dihydroxyvitamin D3 and the serum free 1,25-dihydroxyvitamin D3 concentrations are increased during combined 17 beta-oestradiol and norethisterone acetate therapy for a year. Assuming that the free concentration 1,25-dihydroxyvitamin D3 reflects the biologically active fraction, this rise may in part explain the preventive effect of hormone replacement therapy on osteoporosis.

Capsaicin treatment induces muscarinic hyperreactivity in guinea pig trachea: a warning.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a widely used tool for the depletion of neuropeptides from sensory C-fibres. Upon capsaicin treatment tachykinins are released, resulting in a variety of responses in the airways. We showed that after capsaicin (0.3 microM; 30 min) treatment of guinea pig tracheal smooth muscle preparations, the maximal contraction of the trachea after methacholine stimulation was strongly increased (capsaicin: 1.147 +/- 0.050 g vs. control: 0.717 +/- 0.047 g). This effect was completely nullified after pretreatment with capsazepine (2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3, 4,5-tetrahydro-1H-2benzazepine; a vanilloid receptor antagonist) and YM38336 (a dual tachykinin NK1 and tachykinin NK2 receptor antagonist). Our results serve as a warning against using capsaicin as a putatively clean pharmacological tool to deplete the neuropeptides from pools on the C-fibres because we showed that capsaicin also strongly influences basal mechanisms in tracheal smooth muscle control.

Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis.

Most of the total circulating 1,25-dihydroxyvitamin D (1,25(OH)2D) is bound to plasma proteins, mainly vitamin D-binding protein (DBP) and albumin. Only a small fraction in plasma exists in the free form. It is widely assumed that the non-protein-bound free hormone reflects the biologically active fraction. We describe a dialysis method for the determination of plasma free 1,25(OH)2D which is relatively easy to perform. In this symmetric or "rate" dialysis method, identical samples are placed at both sides of a membrane. At one side, tritiated 1,25(OH)2D is added and the rate of transfer of this tritiated 1,25(OH)2D through a dialysis membrane is directly related to the free fraction of plasma 1,25(OH)2D. This method is much less susceptible toward tracer impurities than indirect equilibrium dialysis and centrifugal ultrafiltration. Moreover, it requires much less tracer. The intraassay coefficient of variation for the determination of the free fraction is 1.0%; the interassay variation is 7.7%. Comparison of the free fraction of 23 samples assessed with both centrifugal ultrafiltration and symmetric dialysis showed much higher values using the former method. No significant correlation between the two methods was found. The free fraction of 1,25(OH)2D in normal subjects as assessed with symmetric dialysis ranges from 0.049 to 0.103%.

The role of prostanoids in ozone-induced changes in airway responsiveness: receptor activation-specific prostanoid release.

We studied the effect of in vivo ozone exposure (3 ppm, 2 h) on methacholine- and histamine-induced guinea pig tracheal smooth muscle contractions in vitro and the role of cyclooxygenase products in this process. After exposure to ozone, methacholine stimulation showed a functional hyperreactivity, whereas after stimulation with histamine a hyporeactivity was observed. These effects could be explained by the release of prostanoids. In a control situation an increase in PGF(2α), PGE(2) and PGD(2) release is observed after stimulation of the histaminergic receptor system. After ozone exposure the release of prostanoids was also enhanced (unstimulated, PGF(2α) and TxB(2); histamine, PGF(2α), PGE(2); methacholine, PGF(2α), TxB(2), 6-kPGF(1α), PGE(2)). This study shows that the prostanoid release is strongly dependent on the receptor system stimulated to induce smooth muscle contraction and the importance of prostanoids in ozone-induced changes in guinea pig tracheal smooth muscle reactivity.

Changes in neuroreceptor function of tracheal smooth muscle following acute ozone exposure of guinea pigs.

We studied the effect of in vivo ozone inhalation (3 ppm, 2 h) on neuroreceptor function in guinea pig tracheal smooth muscle in vitro and the role of the epithelial layer in this process. Changes in smooth muscle tension after stimulation of the muscarinic- and beta-adrenergic receptor were recorded isometrically and stained tracheal tissue sections were histologically evaluated for changes in the epithelial and smooth muscle layer. Ozone exposure resulted in an increase in maximal contraction following stimulation of the muscarinic receptor, whereas pD2 values remained unchanged. After stimulation of the beta-adrenergic receptor no increase in maximal relaxation but only an increase in pD2 value was observed after correction for differences in precontraction level in control- and ozone-exposed situations. Mechanical removal of the epithelial layer resulted in a slight increase of the maximal contraction level after stimulation with methacholine in the control situation, whereas exposure to ozone resulted in a strong decrease of the maximal contraction level under these conditions. Histological stainings showed a slight and focal influx of neutrophilic granulocytes in the epithelial layer, submucosal layer and airway lumen after exposure to ozone. These data support the idea that ozone is able to increase the maximal degree of airway narrowing upon muscarinergic stimulation, i.e. a hyperreactivity response. The results also suggest that functionally altered epithelium plays an important role in the process of ozone-induced hyperreactivity, possibly linked with an early inflammatory response.

Acute exposure to ozone does not influence neuroreceptor density and sensitivity in guinea pig lung.

The effects of acute exposure of guinea pigs to 3 ppm of ozone for 2 h on the receptor density and sensitivity of the muscarinergic-, the histaminergic- and the beta-adrenergic receptor systems were studied, in order to provide more insight in the complex mechanisms underlying the well known ozone-induced changes in receptor functionality. The exposure to ozone did not change either the total amount of receptors present in lung tissue, nor the receptor sensitivity of the systems studied. Although no effects were observed, this does not yet fully exclude the receptor system for being a target of ozone exposure. The receptor function can be changed after exposure to ozone, e.g., the coupling with the G-protein can be influenced. Furthermore, the G-protein itself may have been altered or changes can occur at lower levels in the receptor signal transmission route leading to functional changes after stimulation of the receptor with an agonist.

The effect of ozone exposure on the release of eicosanoids in guinea-pig BAL fluid in relation to cellular damage and inflammation.

The observed effects after ozone exposure strongly depend on ozone concentration and exposure time. We hypothesized that depending on the O3 exposure protocol, mainly either an oxidant damage or an inflammation will determine the O3 toxicity. We compared two different ozone exposure protocols: an acute exposure (3 ppm 2 h) for studying the oxidant damage and an exposure (1 ppm 12 h) where an inflammatory component is also probably involved. We measured LDH activity and protein and albumin exudation as markers for cellular damage. After the acute exposure an increase in LDH activity was measured and after exposure to 1 ppm ozone for 12 h the exudation of protein and albumin was also enhanced. The histological examinations showed a neutrophilic inflammatory response only after exposure to 1 ppm ozone for 12 h. The acute exposure protocol resulted in an increased release of PGE2, PGD2, PGF2alpha and 6-ketoPGF1alpha whereas exposure to 1 ppm ozone for 12 h led to an additional release of LTB4. No effects were measured on the release of TxB2 and LTC4/D4/E4. These changed amounts of eicosanoids will probably contribute to the ozone-induced lung function changes.

Hormone replacement therapy increases serum 1,25-dihydroxyvitamin D: A 2-year prospective study.

Osteoporosis is a common disorder in postmenopausal women, which is probably due to decreased ovarian function. Currently, hormone replacement therapy (HRT), involving administration of estrogen and progestogen, is successfully applied to reduce bone resorption. We studied the effect of HRT on 23 postmenopausal women. This consisted of a combination of 17 beta-estradiol and dydrogesterone, on the serum level of 1,25-dihydroxyvitamin D (1,25(OH)2D) after 0, 6, 12, and 24 months. We found mean serum concentrations (+/- SD) of 1,25(OH)2D of 130.5 pmol/liter (46.1), 152.7 pmol/liter (45.1), 170.8 pmol/liter (64.0), and 155.2 pmol/liter (59.7), respectively. The baseline values in these women were found to be significantly lower than those during therapy (P < or = 0.005). No statistically significant differences were observed when comparing the estrogen-only phase with the combined estrogen-progestogen phase. It is concluded that HRT results in an increase in the serum 1,25(OH)2D concentration which lasts for at least 2 years. This increase may partly explain the preventive effect of HRT on osteoporosis. Furthermore, these results suggest that dydrogesterone does not influence the estrogen-induced changes in serum 1,25(OH)2D concentration.

Induction of Ca2+ oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells.

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca2+ sequestering and releasing properties of internal Ca2+ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca2+ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P3]-sensitive, but not the Ins(1,4,5)P3-insensitive, Ca2+ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca2+ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca2+ concentration ([Ca2+]i,av), which was largely independent of external Ca2+. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonist-evoked [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-ATPase activity resulted in a [Ca2+]i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-sensitive and -insensitive Ca2+ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca2+]i. The U73122-evoked oscillations were abolished in the absence of external Ca2+. The ability of U73122 to generate external Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.

Advantages of paper chromatography as a preparative step in the assay of 1,25-dihydroxyvitamin D.

Ether extraction and paper chromatography were used to separate the main metabolites of vitamin D in plasma [25-(OH), 24,25-(OH)2 and 1,25-(OH)2 vitamin D]prior to radio receptor-assay. The overall procedural loss of the 1,25-(OH)2 vitamin D was 58 +/- 5% (n = 40), corrected for by tracer addition. The sensitivity of the assay was 0.5 fmol/tube, corresponding to 4 pmol/l, and the intra- and inter-assay coefficients of variation were 10.5% and 11.5%, respectively. The range of values measured in healthy controls was 80-200 pmol/l (n = 60), which is in agreement with findings reported in the literature. A comparison of the results of the present procedure with those obtained with a procedure employing C18 purification, disclosed a correlation coefficient of 0.92 (p < or = 0.0001), a slope of 0.89 (p < or = 0.0001) and a small non-significant intercept of 5.0 pmol/l (n = 53).

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state.

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokinin-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 microM staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Low ratio of androstenedione to testosterone in plasma and saliva of hirsute women.

To gain insight into the passage of androstenedione (A4) through the salivary gland, we measured concentrations of plasma total (TA4), free (FA4), and salivary (SA4) androstenedione during administration of dexamethasone and synthetic corticotropin to control subjects and hirsute women and compared these data with plasma total (TT), free (FT), and salivary testosterone (ST) concentrations. TA4 and FA4 were significantly lower in control subjects throughout (0, 15, 45, and 75 min) (P less than 0.05). SA4 in control subjects was significantly lower only in the follicular phase at 0 and 15 min. There was no significant difference between the increments in SA4 in response to corticotropin. The concentration of SA4 in the control women at 0 min was not significantly different from the concentration of FA4. At 45 and 75 min after corticotropin administration, however, SA4 was slightly higher than FA4 (P less than 0.01). In hirsute women, however, the concentration of SA4 was significantly lower than FA4 at all times (P less than 0.05). TT, FT, and ST concentrations were about twofold higher in the hirsute women than in control subjects throughout. In both groups, ST concentrations were three times as high as FT concentrations (P less than 0.001). The SA4:ST ratio was significantly lower than the FA4:FT ratio in both groups (P less than 0.001) because of higher ST than FT concentrations and similar or even lower SA4 concentrations in both groups. Both FA4:FT and SA4:ST ratios were lower in hirsute women, except for the FA4:FT ratio in control subjects in the luteal phase. Our data are compatible with 17-hydroxysteroid oxidoreductase activity in the salivary gland. If the difference in the ratios of A4 to T between hirsute women and control subjects is attributed to this hypothetical enzymatic activity, it would suggest a more rapid conversion of A4 to T in the hirsute group.

Concentrations of salivary testosterone and plasma total, non-sex-hormone-binding globulin-bound, and free testosterone in normal and hirsute women during administration of dexamethasone/synthetic corticotropin.

Preliminary data indicate that the concentration of circulating cortisol may affect the binding of testosterone to plasma proteins. This interdependency was evaluated by the assessment of plasma cortisol, salivary (ST) and plasma total testosterone (TT), testosterone not bound to sex-hormone-binding globulin (n-SHBGT), and free testosterone (FT) in normal and in hyperandrogenemic women during combined dexamethasone/synthetic corticotropin administration. The concentrations of TT, FT, and ST significantly increased (P less than 0.001) during this dynamic test both in the controls and in hirsute women. Remarkably, however, n-SHBGT remained virtually unchanged in normal women in the follicular phase, decreasing in the luteal phase, and decreasing even more markedly in the hirsute women. Both the normal and the hirsute women showed a statistically significant negative correlation between the percentage of n-SHBGT and plasma total cortisol (P less than 0.001). The apparent decrease of n-SHBGT was the result of displacement of T from corticosteroid-binding globulin (CBG) by the increase in cortisol after the infusion of synthetic corticotropin: the CBG-bound T was measured as n-SHBGT because CBG was not precipitated with 50% saturated ammonium sulfate. Our results indicate that ST or FT better represents the clinical status of androgenicity than does n-SHBGT, as assessed by ammonium sulfate precipitation, because n-SHBGT also depends on the concentration of cortisol.