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Biomedical subjects

J Merke

Publications and source records attributed to J Merke.

At least 19 recordsLinked to original sources
PubMed1991

1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat.

Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)2D3 receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [3H]-1,25(OH)2D3 is noted in both the logarithmic growth phase and at confluence (Nmax 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. KD was 10(-11) M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)2D3 receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest Nmax in the proliferating cartilage. 1,25(OH)2D3 in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10(-12) M and dose-dependent inhibition at 10(-10) M and below. Inhibition was specific and not seen with 24,25(OH)2D3 or dexamethasone. Growth phase-dependent 1,25(OH)2D3 receptor expression and effects of 1,25(OH)2D3 on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.

Alkaline Phosphatase↗
PubMed1991

Actions of 1,25-dihydroxyvitamin D3 on human mesangial cells.

In the present study, we examined specific binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by an effects of 1,25(OH)2D3 on human mesangial cells (hMC), obtained from healthy portions of tumor-bearing kidneys. Receptors for 1,25(OH)2D3 were characterized by (1) sucrose density gradient analysis, (2) Scatchard analysis, and (3) DNA affinity of the receptor molecule. Specific binding occurred by a single class of macromolecules, sedimenting with 3.5 S in sucrose density gradients (5% to 20%). Receptors showed high affinity (Kd, 1.39 x 10(-10)), and specific binding capacity (Nmax) of 821 binding sites per cell. 1,25(OH)2D3 (10(-6) to 10(-10)) reduced both DNA synthesis (by [3H]thymidine incorporation) and cell growth (by cell counting) throughout the log-phase and confluence. Further evidence of functional effects of 1,25(OH)2D3 on hMC is provided by ultrastructural studies, which showed rapid increase of electron-dense lysosomal particles in hMC exposed to 1,25(OH)2D3. The data identify actions of 1,25(OH)2D3, a molecule with recently recognized immunoregulatory roles, on hMC. The results are consistent with a role of 1,25(OH)2D3 in control of mesangial cell function.

Calcitriol↗
PubMed1991

Effects of 1,25(OH)2D3 on compensatory renal growth in the growing rat.

Renal compensatory growth after uninephrectomy (UNX) was examined in vitamin D replete male 100 g Sprague-Dawley rats. Five days after UNX, the contralateral kidney wet weight increased by 25% with the kidney weight/body weight ratio reaching a plateau by day 7 after UNX. The early weight increase was primarily due to an increased cell number, as evaluated by a stereological technique in perfusion-fixed kidneys. Twenty pmol 1,25(OH)2D3 by daily s.c. injection increased time-averaged 1,25(OH)2D3 concentrations 3.3-fold and reduced the increment in the kidney weight of UNX pairfed rats compared to solvent UNX controls. The number of mitoses (whole kidney and different nephron segments) were significantly reduced by giving 1,25(OH)2D3 to UNX animals at different levels of food intake. The effect was also demonstrable in PTX animals on a constant infusion of exogenous PTH (100 ng/kg/hr 1,34 bPTH by osmotic minipump). The data suggest that changes of 1,25(OH)2D3 concentration within a physiologically relevant range modulate compensatory (and possibly basal) growth of the kidney.

Animals↗
PubMed1991

Hyperparathyroidism and abnormal 1,25(OH)2 vitamin D3 metabolism in experimental lead intoxication.

Clinical evidence points to disturbed calcium metabolism in lead (Pb) intoxication. To further clarify the mechanisms involved, serum levels of 1,25(OH)2D3, receptors for 1,25(OH)2D3 as well as size and ultrastructure of parathyroid glands were examined in Wistar Kyoto rats exposed to 1% lead (Pb) acetate in drinking water for 10 weeks (short-term study) or 0.001-1% Pb acetate for 24 weeks (long-term study). After administration of Pb for 10 weeks, bone Pb was significantly increased (641 +/- 66.9 (SD) vs. 0.648 +/- 0.39 mg kg-1 ash in controls). Total serum calcium and ionized Ca2+ (1.15 +/- 0.031 vs. 1.25 +/- 0.03 mmol l-1) were significantly decreased. Renal function (Ccr) was unchanged, but urinary cAMP excretion and circulating 1,25(OH)2D3 (177 +/- 10.9 vs. 232 +/- 18.9 pmol l-1) were diminished. Specific binding of 1,25(OH)2D3 was increased in parathyroids (Bmax 128 +/- 4.7 vs. 108 +/- 0.6 fmol mg-1 protein) and intestinal muscosa; Bmax failed to adequately rise in response to pretreatment with 1,25(OH)2D3 (2 x 10 ng day-1 for 4 d) in Pb-exposed animals. Receptor characteristics (sedimentation constant, KD, DNA affinity) were unchanged. Parathyroid weight was significantly increased (178 +/- 25 vs. 96 +/- 34 micrograms) with no change of estimated nuclear volume, cell volume or cell ultrastructure. After 24 weeks of Pb exposure, a dose-dependent but non-linear increase of parathyroid weight was noted between 0.001% and 1% Pb in drinking fluid. The present study documents secondary hyperparathyroidism associated with, and presumably caused by, hypocalcaemia and low 1,25(OH)2D3 levels, in experimental Pb intoxication.

Animals↗
PubMed1991

No decrease of 1,25(OH)2D3 receptors and duodenal calbindin-D9k in uraemic rats.

In parathyroids of uraemic patients or animals, decreased specific binding of 1,25(OH)2D3 has been observed and implicated in the genesis of secondary hyperparathyroidism of renal failure. We re-examined binding of 1,25(OH)2D3 using chromatin preparations for receptor characterization which differed from previous studies (a) by inclusion of protease inhibitors (PMSF, aprotinin) and molybdate in the extraction buffer and (b) by omitting the K-extraction step. With this method, the Nmax in the intestinal mucosa and parathyroids of uraemic animals was significantly higher, while the receptor sedimentation constant (S), DNA affinity and KD were all unchanged. The ratio of occupied to total receptors was not significantly altered. The regulation of 1,25(OH)2D3 receptors in response to acute injection of 1,25(OH)2D3 was abnormal. Calbindin-D9k concentration in the intestines of uraemic and control rats was comparable both before and after administration of 1,25(OH)2D3. The present data demonstrate (a) increased 1,25(OH)2D3 receptors and (b) unchanged 1,25(OH)2D3-dependent synthesis of calcium binding protein (CaBP) in experimental uraemia.

Animals↗
PubMed1989

Evidence for in vivo upregulation of 1,25(OH)2 vitamin D3 receptor in human monocytes.

Mammalian cells increase net expression of 1,25(OH) 2D3 receptors after exposure to physiological concentrations of 1,25(OH) 2D3 in vitro. We examined specific binding of 1,25(OH) 2D3 by human monocytes before and after daily administration of 1.5-2 micrograms 1,25(OH) 2D3 p.o. for 3 days in 5 healthy normal D-replete probands. Median specific binding (Nmax) at baseline was 793 molecules/cell and 2052 or 2828 at 24h and 72h of 1,25(OH) 2D3 treatment respectively. The results suggest (a) upregulation of 1,25(OH) 2D3 receptors occurs in man and (b) monocyte preparations can be used to assess receptor regulation in vivo.

Adult↗
PubMed1989

1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia.

Parathyroid cell proliferation and parathyroid hyperplasia are features of renal secondary hyperparathyroidism. Since parathyroids have recently been recognized as an important target for 1,25(OH)2D3, the effects of administration of variable doses of 1,25(OH)2D3 on ex vivo radiothymidine incorporation in the parathyroid glands, on parathyroid cell mitoses, on parathyroid weight, morphometric indices and on parathyroid protein/DNA ratio were examined in rats with uremia (subtotal nephrectomy; NX) or with calcium deficiency. 3H-thymidine incorporation (3 hr; 37 degrees C; PBS with 10 mmol glucose) was elevated in NX animals, that is, 204 +/- 51 dpm/micrograms DNA versus 96 +/- 28 in controls. In vivo pretreatment with 1,25(OH)2D3, either by intermittent i.p. injection or by osmotic minipump, dose-dependently decreased 3H-thymidine incorporation and parathyroid cell mitoses without affecting morphometric indices of parathyroid cells. Prophylactic administration (i.p.) of 1,25(OH)2D3, starting on the day of nephrectomy, prevented parathyroid hyperplasia (NX + 1,25(OH)2D3 0.84 micrograms tissue/g body wt vs. 1.25 micrograms in untreated NX and 0.54 in ad libitum fed controls), but 10 days of treatment beginning on the 21st day of uremia did not reverse existing hyperplasia (NX + 1,25(OH)2D3 1.5 micrograms/g body wt vs. 1.37 micrograms in untreated NX and 0.56 micrograms in ad libitum fed controls). The inhibitory effect was specific for 1,25(OH)2D3 and not imitated by Dexamethason. However, the effect was not specific for parathyroid hyperplasia of uremia, since similar inhibition of 3H-thymidine incorporation by 1,25(OH)2D3 was also observed in rats on low calcium diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Hyperparathyroidism and abnormal calcitriol metabolism in the spontaneously hypertensive rat.

Abnormalities of calcium metabolism and of its two principal regulating hormones, parathyroid hormone and 1,25-dihydroxyvitamin D3 (calcitriol), have been reported in the spontaneously hypertensive rat (SHR). Reports of abnormal calcitriol metabolism in the SHR by several groups have not provided measurements of tissue calcitriol receptors. Similarly, few data are available as to the parathyroid status of the SHR. In the present study, circulating calcitriol levels and intestinal and parathyroid gland calcitriol receptor status were determined in male SHR and in Wistar-Kyoto (WKY) rats. Parathyroid status was investigated by determination of parathyroid gland mass together with tissue micromorphometry and by quantitative histology of bone as a measure of the biological action of parathyroid hormone. Circulating calcitriol levels were reduced in the 11-week-old SHR compared with the WKY rat (165 +/- 23 vs. 194 +/- 28 pmol/l, p less than 0.01, mean +/- SD). Calcitriol-free ratio was diminished and maximal specific binding capacity for calcitriol was increased in the SHR in parathyroid tissue (172 +/- 4.9 vs. 123 +/- 6.6 fmol/mg protein, p less than 0.01) and in intestinal mucosa with no change of receptor affinity. Plasma ionized calcium (1.29 +/- 0.05 vs. 1.45 +/- 0.35 mmol/l, p less than 0.05) and phosphate (1.5 +/- 0.26 vs. 2.4 +/- 0.03 mmol/l, p less than 0.05) were significantly lower in the SHR. Parathyroid gland mass was increased in the SHR (59 +/- 12 vs. 17 +/- 7 micrograms/100 g body wt, p less than 0.001) as a result of hyperplasia and not hypertrophy. Higher osteoclast numbers were observed in SHR bone (27.6 +/- 0.79 vs. 23.9 +/- 0.66 osteoclasts/mm2, p less than 0.01), suggesting increased parathyroid hormone activity. In summary, in the 11-week-old SHR we observed reduced circulating calcitriol levels together with increased tissue calcitriol receptor numbers, increased parathyroid gland mass, and histological evidence of hyperparathyroidism. It is possible that these abnormalities influence the development of hypertension in the SHR.

Animals↗
PubMed1989

Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

Animals↗
PubMed1989

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Animals↗
PubMed1987

Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cells in vitro.

Binding of [3H] 1,25(OH)2D3 and effects of 1,25(OH)2D3 on cell ultrastructure were evaluated in vascular smooth muscle cells (VSMC) primary cultures (aortic media). Specific reversible binding of [3H] 1,25(OH)2D3 by a 3.5 S macromolecule with DNA binding, KD 6.2 X 10(-10) M and Nmax 16 fmol/mg protein was demonstrated. Incubation of VSMC with 10(-8) M 1,25(OH)2D3, but not 25(OH)D3, in the presence of 10% FCS for up to three weeks caused rapid reversible appearance in the cytoplasm of membrane-bounded electron-dense lysosomal particles which on electronspectroscopic imaging contained Ca and Pi. VSMC are targets for vitamin D.

Animals↗
PubMed1987

1,25(OH)2D3 receptors and endorgan response in experimental aluminium intoxication.

Severe aluminium-induced osteomalacia is refractory to treatment with 1,25(OH)2D3 which frequently causes hypercalcemia. To further explore the mechanisms involved, we have utilized a model of short-term aluminium intoxication in the rat (total: 11 mg elemental aluminium in 3 weeks) to study (a) 1,25(OH)2D3 receptor status in a variety of classical and non-classical target organs for 1,25(OH)2D3; (b) circulating 1,25(OH)2D3 levels; (c) baseline duodenal calcium transport, utilising the Ussing chamber, to investigate the functional significance of receptor status in a classical target organ; and (d) duodenal calcium transport response to exogenously administered 1,25(OH)2D3. Both in the three week model and in the 16 week model (total: 41 mg elemental calcium) increased maximal specific binding capacity for 1,25(OH)2D3 (Nmax), that is, number of unoccupied receptors, was observed in nuclear fractions of all tissues studied. Receptor affinity, the apparent dissociation constant KD, was unchanged. Total binding capacity, measured after displacement of endogenous ligand by Mersalyl, that is, the sum of occupied plus non-occupied receptors, was also increased. Both circulating 1,25(OH)2D3, mucosa-to-serosa calcium flux (Jms) and net calcium flux (Jnet) were reduced under baseline conditions, suggesting the lack of a direct relationship between receptor expression and endorgan response. Following exogenous 1,25(OH)2D3 administration, calcium Jms and Jnet were significantly lower in the aluminium intoxicated animals, with the increment induced in Jnet in aluminium intoxicated animals being 63% of that induced in controls. Our data suggest that resistance to the action of 1,25(OH)2D3 in aluminium intoxication is postreceptor in nature.

Aluminum↗
PubMed1987

Diminished parathyroid 1,25(OH)2D3 receptors in experimental uremia.

In Sprague Dawley rats, six days after subtotal nephrectomy, serum 1,25(OH)2D3 concentration was diminished (59.8 +/- 17.5 pg/ml vs. 121 +/- 48; P less than 0.01). Despite low circulating 1,25(OH)2D3 levels, maximal specific binding capacity for 1,25(OH)2D3 in parathyroid glands was diminished (Nmax 87.5 fmol/mg protein and 3.52 fmol/mg DNA vs. 143 fmol/mg protein and 4.75 fmol/mg DNA, respectively). There was no change of KD, apparent molecular size (sucrose density gradient) and DNA binding affinity (DNA cellulose chromatography) pointing to intactness of the receptor. Since 1,25(OH)2D3 is a potent negative feedback signal for parathyroids, the data are potentially relevant for the genesis of secondary renal hyperparathyroidism.

Animals↗