Zur antimikrobiellen Wirkung von Vitamin D3

Zusammenfassung Bei 5 überprüften Bakterienarten wurde ein wachstumshemmender Effekt von Vitamin D₃ festgestellt. Dabei ließ sich oberhalb von 5×10⁴ bis 9×10⁴ IE/ml Vitamin D₃ ein deutlicher Wachstumsabfall bis hin zur völligen Bakterizidie ermitteln.Den Herren Professoren Dr. W. Döll und Dr. H. Seeliger (Institut für Hygiene und Mikrobiologie der Universität Würzburg) sei für liebenswürdige Unterstützung gedankt     

Renal handling of calcium and phosphate

Summary In glomerular filtrate, calcium concentration (UF) is some 50–70% of total plasma calcium concentration. About 60% of filtered calcium is reabsorbed in the proximal convoluted tubule, some 15% in pars recta, 15% in the thick ascending limb, and 9% in the distal convoluted tubule including the granular portion of the cortical collecting duct. About 1% is excreted. Approximately 2/3 of proximal tubular calcium transport is passive, driven by a chemical (TF/UF=1.1) and electrical (2 mV, lumen positive) gradient; 1/3 is active, whereby calcium enters the cell at the brush border membrane by passive diffusion and is pumped out at the basolateral membrane in exchange for sodium. Transport in distal convoluted tubules is entirely active and operates both against a chemical (TF/UF 0.3–0.7) and electrical (10–70 mV, lumen negative) gradient. Although saturability of transport is not detectable in microperfusion studies, enhancement of plasma calcium leads to calciuria. Factors stimulating calcium reabsorption are parathyrin, 1.25(OH)₂D₃, thiazides, and alkalosis. Factors inhibiting calcium reabsorption are calcitonin, growth hormone, thyroid hormone, chronic application of mineralo- and glucocorticoids, insulin, glucose, acidosis, sodium infusion, acetazolamide, furosemide, and mannitol.Phosphate concentration in ultrafiltrate is some 90% of total plasma concentration. In intact animals about 2/3 of filtered phosphate is reabsorbed in proximal convoluted tubules, and some 20% is excreted in urine. Shortly after thyroparathreoidectomy (TPTX), 80% of filtered phosphate is reabsorbed in the proximal convoluted tubule, 15% in the pars recta, and 2% is excreted in the urine. Thus, less phosphate is excreted in urine (20% in intact, 2% in TPTX animals) as is recovered in late distal convoluted tubules of superficial nephrons (35% and 5%, respectively). The discrepancy is at least partially due to more avid reabsorption in deep nephrons. Furthermore, evidence exists in favor of phosphate reabsorption in the arcades or collecting duct. Phosphate reabsorption in proximal convoluted tubule and pars recta is active. Uptake of phosphate at the brush border membrane is uphill and is driven by coupling with two sodium ions, whereas exit at the basolateral membrane may be entirely passive. At increasing plasma phosphate concentrations, reabsorption is saturated and excess filtered phosphate excreted. Factors stimulating phosphate transport are phosphate depletion, acute 1,25(OH)₂D₃, thyroxin, growth hormone, magnesium, lithium, and metabolic acidosis. Factors inhibiting phosphate transport are high phosphate or high calcium diets, parathyrin, calcitonin, chronic 1,25(OH)₂D₃, hypocalcemia, magnesium deficiency, respiratory acidosis, metabolic alkalosis, glucose, colchicine, NaCl-infusions, thiazides, furosemide, ethacrynic acid, mannitol, and acetazolamide.     

Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: Phosphate transport

The polarized distribution of phosphate (P_i) transport systems in a continuous renal cell line derived from opossum kidney (OK) was measured in monolayers grown on permeant filter support. When cultured on collagen-coated nitrocellulose filters, OK cells formed tight, functionally polarized monolayers. Three P_i transport systems were identified in these monolayers: one apical sodium (Na)-dependent system and two systems on the basolateral surface, one Na-dependent and one Na-independent. The apical system was high-affinity (K _m=0.4 mM P_i), low-capacity (J _max=1100 pmol P_i/mg protein per minute) with a NaP_i stoichiometry greater than 1 (n=3) and a high interaction coefficient (K ^Na=105 mM Na). On the basolateral surface the Na-independent system comprised about 30% of the total P_i transport at this surface. Both basolateral systems were of low affinity (K _mNa-independent, 2.6 mM; Na-dependent, 5.2 mM) and high capacity (J _maxNa-independent, 2100; Na-dependent, 2400 pmol/mg protein per minute). The basolateral Na-dependent system had a Na_i stoichiometry of 1 and a relatively low interaction coefficient (K ^Na=25 mM Na). Only the basolateral Na-independent system was inhibitable by 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These results are compatible with a net vectorial transcellular transport of P_i from the apical through the basolateral cell surfaces. The presence of a basolateral Na-dependent system may reflect additional metabolic requirements that cannot be met only by apical influx. Taken together, these results demonstrate the ability to grow cell monolayers successfully, displaying polarized transport activities similar to in situ.     

Vitamin D Enhances Neutrophil Generation and Function in Zebrafish (Danio rerio)

Vitamin D (VD) is a major regulator of calcium metabolism in many living organisms. In addition, VD plays a key role in regulating innate and adaptive immunity in vertebrates. Neutrophils constitute an important part of the first line of defense against invading microbes; however, the potential effect of VD on neutrophils remains elusive. Thus, in this study zebrafish in different developmental stages were utilized to identify the potential role of VD in the basal homeostasis and functions of neutrophils. Our results showed that addition of exogenous VD₃ promoted granulopoiesis in zebrafish larvae. Reciprocally, neutrophil abundance in the intestine of adult zebrafish with a cyp2r1 mutant, lacking the capacity to 25-hydroxylate VD, was reduced. Moreover, VD-mediated granulopoiesis was still observed in gnotobiotic zebrafish larvae, indicating that VD regulates neutrophil generation independent of the microbiota during early development. In contrast, VD was incapable to influence granulopoiesis in adult zebrafish when the commensal bacteria were depleted by antibiotic treatment, suggesting that VD might modulate neutrophil activity via different mechanisms depending on the developmental stage. In addition, we found that VD₃ augmented the expression of il-8 and neutrophil recruitment to the site of caudal fin amputation. Finally, VD₃ treatment significantly decreased bacterial counts and mortality in zebrafish infected with Edwardsiella tarda (E. tarda) in a neutrophil-dependent manner. Combined, these findings demonstrate that VD regulates granulopoiesis and neutrophil function in zebrafish immunity.     

Phosphate fluxes in isolated enterocytes from vitamin D replete and vitamin D deficient rats — early effects of calcitriol

In the present work we studied rapid in vitro effects of calcitriol (1,25(OH)₂ vitamin D₃) on the intestinal transport of inorganic phosphate (P_i). Enterocytes from vitamin D replete (D⁺) as well as vitamin D depleted (D^–) rats were isolated mechanically from the duodeno-jejunum. In this model, P_i uptake was a temperature and Na⁺-dependent phenomenon. The in vitro-addition of calcitriol (1 pM) resulted in a significant enhancement of initial P_i uptake rate by enterocytes from D⁺ (P<0.01) and D^– (P<0.05) rats. This effect which was Na⁺-dependent, was observed within the time of 20 min, but not before. A similar effect on P_i uptake rates of D⁺ or D^– enterocytes could be elicited by the in vitro addition of the methyl ester of cis-vaccinic acid (MCVA) which is thought to increase membrane fluidity by modifying the lipid composition of the cell membrane. The stimulatory effect of calcitriol on P_i uptake rate was blunted in the presence of the methyl ester of transvaccinic acid (MTVA) thought to decrease membrane fluidity. Enterocyte P_i efflux rate constant (^o KP_i) remained unchanged in the presence of calcitriol (1 pM). In conclusion, the study demonstrates a rapid in vitro effect of calcitriol on P_i uptake by isolated enterocytes from D⁺ and D^– rats. It suggests, but does not prove, that the hormone may act via an action independent of genomic nuclear activation.     

Lack of involvement of sarcoplasmic reticulum in myopathy of acute phosphorous depletion

Summary Acute and chronic hypophosphatemia are known to cause metabolic myopathy. It has been proposed that impaired Ca transport in subcellular membranes is involved in its genesis. In the present study, calcium transport in the sarcoplasmic reticulum (SR), concentrations of ions or nucleotides and transmembrane potential were investigated in muscles of acutely hypophosphatemic rats, i.e. animals with chronic dietary phosphorous deprivation (PD) and superimposed acute hypophosphatemia resulting from the administration of insulin and glucose. Despite hypophosphatemia and low muscle phosphorous concentration, no significant change of the initial rate of Ca uptake or Ca concentrating ability was observed in the SR of PD rats. Storing capacity was decreased; this may result from altered vesicle geometry. Water content, Na concentration, the concentration of several nucleotides and transmembrane potential of muscle were unchanged in PD rats. The findings document that no intrinsic abnormality of vectorial Ca transport is present in the SR of acutely hypophosphatemic PD animals.Visiting investigator with scholarship of Humboldt-Stiftung (Universidad Nacional del Sur, Departamento de Ciencias Naturales, 800 Bahia/Argentina)     

Disturbed calcium metabolism in subjects with elevated diastolic blood pressure

Summary Essential hypertension has been associated with disturbed calcium metabolism, but the available data are controversial. We measured parameters of calcium metabolism in groups of untreated male subjects (n = 78) with elevated diastolic blood pressure (101 ± 6 mmHg, mean ± SD) and age-matched male subjects (n=79) with low diastolic blood pressure (62 ± 4 mmHg). The participants of the study were drawn from a random population sample. Subjects with high diastolic blood pressure had significantly higher carboxy-terminal parathyroid hormone (PTH) plasma concentrations than controls with low diastolic blood pressure (median 114 vs. 43 pmol/l, P < 0.01). The 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentrations were comparable in both groups. Individuals with high diastolic blood pressure had significantly lower total serum calcium (2.41 ± 0.10 vs. 2.47 ± 0.10 mmol/l, mean ± SD; P < 0.01). PTH concentrations were correlated with diastolic pressure (r = –0.39, P < 0.001). The data are compatible with increased parathyroid activity despite unchanged concentrations of vitamin D metabolites in human hypertension.Abbreviations PTH parathyroid hormone - C-PTH carboxy-terminal parathyroid hormone - 1,25(OH)₂D 1,25-di-hydroxyvitamin D - 25(OH)D 25-hydroxyvitamin D     

Different inflammatory reactions to vitamin D3 among the lateral, third and fourth ventricular choroid plexuses of the rat

The four choroid plexuses in the brain ventricles are not identical, but differences among them have rarely been studied. The present work concerns the inflammatory and hemorrhagic choroid plexitis produced in Lewis rats by a single gavage of cholecalciferol (vitamin D₃) or related steroids with vitamin D activity. Plexitis was very severe in the fourth ventricular plexus, somewhat less severe in the lateral ventricular plexuses, and almost absent in the third ventricular plexus. These findings were compared to the scanty data from the literature on differences among the plexuses.     

Regulation of phosphate transport in proximal tubules

Homeostasis of inorganic phosphate (P_i) is primarily an affair of the kidneys. Reabsorption of the bulk of filtered P_i occurs along the renal proximal tubule and is initiated by apically localized Na⁺-dependent P_i cotransporters. Tubular P_i reabsorption and therefore renal excretion of P_i is controlled by a number of hormones, including phosphatonins, and metabolic factors. In most cases, regulation of P_i reabsorption is achieved by changing the apical abundance of Na⁺/Pi cotransporters. The regulatory mechanisms involve various signaling pathways and a number of proteins that interact with Na⁺/P_i cotransporters.     

Glutathione-dependent inactivation of sodium-dependent phosphate transport across rat renal brush-border membrane

Thiol/disulfide is fundamental in protein function; we previously observed an inhibitory effect of thiol oxidants on the Na-dependent phosphate (Pi) uptake into renal brush border membrane vesicles (BBMV). We examined whether oxidation of glutathione (GSH) is involved in the mechanism. Vesicular thiols were measured by liquid chromatography. BBMV were incubated with reagents before an influx of Pi. Diamide (5 mM) reduced the capacity of the Pi uptake. Subsequent treatment with dithiothreitol (5 mM) blocked the inhibitory effect of diamide. Vesicular GSH was not modified only by the incubation, whereas it was oxidized by the treatment with diamide, and reduced by dithiothreitol. Furthermore, in vivo treatment with cAMP provided GSH-depleted BBMV without any influence on Pi uptake. Diamide did not inhibit the transport of Pi into GSH-depleted vesicles, but it did inhibit the uptake when GSH was introduced into the vesicles. In conclusion, a GSH-dependent mechanism is involved in the inhibitory effect of diamide on sodium-dependent Pi transport across the renal brush-border membrane.     

Unidirectional influx of phosphate across the mucosal membrane of rabbit small intestine

The influx of phosphate across the mucosal border of different regions of rabbit small intestine was investigated using the technique of Schultz et al., J. Gen. Physiol.50, 1241–1260 (1967). In the duodenum, the phosphate influx consisted of two components: 1. a saturable part, inhibited competitively by the presence of arsenate in the mucosal solution and strongly dependent on the mucosal Na concentration, and 2. a Na-independent part, linearly related to the mucosal phosphate concentration. In the jejunum and the ileum, the phosphate influx was a linear function of the mucosal phosphate concentration. In these regions arsenate had no effect on the influx, supporting the idea of a diffusional transport. HgCl₂ (0.5 mM) reduced the phosphate influx in the duodenum, at 140 mM Na, to the levels under Na-free conditions. The Na-independent influx was only slightly decreased by HgCl₂, suggesting that this agent affects mainly the Na-dependent phosphate influx. In the ileum HgCl₂ decreased the influx by about the same amount as under Na-free conditions in the duodenum. Thus, in the rabbit, the duodenum only appears to have a Na-dependent, carrier mediated phosphate transport mechanism at the mucosal membrane. In the jejunum and the ileum the phosphate uptake seems to be by simple diffusion.Presented in part at the Second Meeting of the European Intestinal Transport Group, Pont-à-Mousson, France, September 6–9, 1978. Abstract published in Gastroenterol. Clin. Biol.3, 173 (1979)     

Phosphate transport in the proximal convolution of the rat kidney

Proximal inorganic phosphate (P _i ) transport was evaluated using the standing droplet method with simultaneous microperfusion of the peritubular blood capillaries.In chronic parathyroidectomized (PTX) rats addition of 3 M of the Ca²⁺ ionophore A 23187 to the luminal perfusate had no effect on the P _i transport, although the isotonic fluid reabsorption was reduced by 20%. When the Ca²⁺ concentration in the perfusates was raised from 1.5 mM to 3.0 mM the reabsorption did not change significantly. But when Ca²⁺ was omitted from the perfusates the P _i reabsorption dropped by 19%, and when 2 mM EDTA were added to the perfusates P _i transport decreased by 35%.The influx of P _i from the interstitial space and from the cell into the phosphate-free luminal perfusate did not change, when the perfusates were Ca²⁺-free, but it increased by 23% in the presence of 2 mM EDTA.The data indicate that 1. a rise in intracellular Ca²⁺ above normal is not a factor which modifies basal P _i transport i.e. when P _i transport is independent of the action of parathyroid hormone. 2. A reduction of extracellular Ca²⁺ concentration from normal toward zero reduces P _i transport without changing the paracellular leak permeability for P _i . 3. With EDTA the paracellular leak permeability for P _i is increased, thus causing an even greater reduction in net P _i transport than with Ca²⁺-free solutions alone.     

Identification of potential components of the transport mechanism for Na+ in the hen colon and coprodaeum

The binding of³H-benzamil to homogenates of epithelium removed from the colon and coprodaeum of hens was studied. A low capacity high affinity binding component was detected. The binding constants for benzamil and amiloride to this component were similar to those obtained when these ligands were used to inhibit transport in intact epithelia. The mean potency ratio of benzamil to amiloride was 12.8 measured by binding compared with 11.6 from functional inhibition. Binding activity was present in tissues taken from animals fed on low sodium diets and those containing a normal sodium content. In the presence of sodium the affinity of benzamil was slightly reduced, but only in tissues taken from animals on a low salt diet. Only a small fraction of the total binding activity was present in the apical surface of low salt tissues indicating that in homogenates a small percentage of the total activity is associated with functional sodium entry sites. It is suggested that the major part of the binding activity detected in homogenates represents components of the sodium ion translocation mechanism which are en route for the apical membrane.     

Long-term therapy with cyclosporin A does not influence serum concentrations of vitamin D metabolites in patients with multiple sclerosis

Summary Animal studies have shown that cyclosporin A (CyA) stimulates renal 25-hydroxyvitamin D₃ [25(OH)D₃]-1-hydroxylase activity; in contrast, studies in renal transplant recipients indirectly suggest that CyA reduces 1,25-dihydroxyvitamin D₃ [1,25 (OH)₂D₃] production. To clarify the effect of CyA on vitamin D metabolite concentrations, we measured parameters of calcium metabolism in 37 CyA-treated patients (median trough whole blood levels 171–222 ng/ml) with multiple sclerosis and initially normal kidney function. The patients participated in a randomized double-blind study to assess the efficacy of CyA in multiple sclerosis. An age- and sex-matched control group (n = 39) received azathioprine (Aza). Measurements were made at the end of a 2-year treatment period. The 1,25(OH)₂D₃ serum concentrations were not significantly different between the two groups, although they were numerically lower in CyA-treated patients [median (range), 28.4 pg/ml (7.8–85.9) vs 41.0 pg/ml (9.2–105.1) in Aza-treated patients]. The 25(OH)D₃ levels were comparable in both groups. There was no correlation between the 25(OH)D₃ and 1,25(OH)₂D₃ concentrations. The renal function in both groups was stable in the last 6 months of the study. At the end of the study period, the endogenous creatinine clearance was significantly lower in the CyA-treated group (85 ± 17 ml/min versus 99 ± 22 in the Aza-treated group, P < 0.05). The carboxyterminal parathyroid hormone (C-PTH) was within the normal range in both groups, although CyA-treated patients had significantly higher concentrations (P<0.01). The urinary excretion of mineral ions, cations and protein was similar in both groups. Our data suggest that long-term treatment with CyA does not cause clinically important alterations of vitamin D metabolism in humans. Subtle differences in the concentrations of 1,25(OH)₂D₃ and C-PTH between CyA- and Aza-treated patients result presumably from a slight impairment of renal function through CyA.Abbreviations CyA cyclosporin A - Aza azathioprine - 25(OH)D₃ 25-hydroxyvitamin D₃ - 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - PTH parathyroid hormone - C-PTH carboxyterminal-PTH - AP alkaline phosphatase - Ccr endogenous creatinine clearance - gamma-GT gamma-glutamyltransferase     

Regulatory role of 1, 25-dihydroxyvitamin D3 and vitamin D receptor gene variants on intracellular granzyme A expression in pulmonary tuberculosis

Vitamin D receptor (VDR) genotypes have been shown to be associated with differential susceptibility or resistance to tuberculosis. The influence of FokI, BsmI, ApaI and TaqI variants of VDR gene on 1, 25(OH)₂ D₃ modulated granzyme A expression of cytotoxic lymphocytes induced by culture filtrate antigen (CFA) of Mycobacterium tuberculosis was studied in 40 pulmonary tuberculosis (PTB) patients and 49 normal healthy subjects (NHS) by flow cytometry. In both the study groups, addition of 1, 25(OH)₂ D₃ (10^− 7M) significantly reduced the percentage of granzyme A positive cells in both unstimulated (NHS, p < 0.0001; PTB, p = 0.02) and stimulated culture conditions (CFA, NHS, p < 0.0001; PTB, p = 0.0001) which correlated positively with the IFN-γ levels (unstimulated, p = 0.01; CFA stimulated, p = 0.004) in NHS. The ApaI aa genotype and bbaaTT extended genotype were associated with a significantly decreased percentage of granzyme A positive cells in NHS (p < 0.05). Our results suggest that 1, 25(OH)₂ D₃ suppresses granzyme A probably by down-regulating Th1 cytokine response. Moreover, the VDR gene variants might regulate cytotoxic T-cell response via 1, 25(OH)₂ D₃ mediated suppression of granzyme A expression in tuberculosis.     

Acid-base maneuvers and phosphate transport in the isolated rat kidney

The influence of bicarbonate (HCO ₃ ^– ), the carbon dioxide tension ( , and pH on phosphate (P_i) excretion were assessed in isolated rat kidneys, perfused in vitro with recirculating synthetic solutions. After establishing control values, the perfusate HCO ₃ ^– or was altered separately. When the perfusate pH was decreased, either by increasing or decreasing HCO ₃ ^– , the absolute and fractional P_i excretions increased. Perfusate alkalinization by slightly decreasing the did not affect P_i excretion, but increasing the perfusate pH with addition of HCO ₃ ^– elicited phosphaturia. Other kidneys were perfused with a solution from which the CO₂/HCO ₃ ^– buffer system was nominally absent. Alkalinization of HCO ₃ ^– -free perfusate had no effect upon P_i excretion, but acidification resulted in marked phosphaturia. Perfusate acidification, whether achieved by decreasing HCO ₃ ^– , increasing , or by adding hydrogen ions, uniformly elicited phosphaturia. The data indicate that either decreasing the extracellular pH or increasing the extracellular HCO ₃ ^– inhibits renal P_i reabsorption.Supported in part by grants from the National Institutes of Health (HL-22836), the Kroc Foundation, and the National Kidney Foundation of Wisconsin     

Intravesicular NAD has no effect on sodium-dependent phosphate transport in isolated renal brush border membrane vesicles

The effects of intravesicular NAD on Na⁺-dependent³²P_i uptake were investigated in isolated rat kidney brush border membrane vesicles (BBMV). NAD was introduced into the vesicles by osmotic shock, and extravesicular NAD was removed by passing the vesicles through a anion exchange column. The effectiveness of the osmotic shock procedure and the hydrolysis of extra- and intravesicular NAD were controlled by enzymatic analysis and thin layer chromatography. ADP-ribosylation of the membrane proteins was analyzed in vesicles osmotically shocked in the presence of either [adenylate-³²P]-NAD or [adenine-2,8-³H]-NAD by SDS-polyacrylamide gel electrophoresis.It was found that the Na⁺-dependent P_i uptake was inhibited when the BBMV were incubated with NAD at alkaline pH, which resulted in rapid NAD hydrolysis. When NAD was present in the intravesicular space only, the Na⁺-dependent P_i uptake was not inhibited.³²P from NAD was rapidly incorporated into a number of brush border membrane proteins, but no incorporation of³H-adenine could be detected.The results provide evidence that NAD does not inhibit P_i transport by a direct interaction with the cytoplasmic side of the brush border membrane. No evidence of ADP-ribosylation of the brush border membrane protein(s) was found.     

Adaptation of phosphate transport to low phosphate diet in renal and intestinal brush border membrane vesicles: influence of sodium and pH

The possible role of changes in the sodium (Na) affinity of the carrier for inorganic phosphate (P_i) in the adaptation of P_i transport to low P_i diet was examined in both renal and intestinal brush border membranes vesicles (BBMV) obtained from the same animal. This role was assessed by measuring the Na concentration resulting in half maximal activation of P_i transport (K _0.5Na) in renal and intestinal BBMV prepared from animals adapted to either low (LPD) or high (HPD) phosphorus diet for 7 days. TheK _0.5Na was not modified by dietary P_i, in both renal and intestinal BBMV. LPD increased maximal P_i transport from 1794.8±198.0 to 296.4±362.0 in renal and from 28.2±3.4 to 80.5±7.2 pmol/mg 10 s in intestinal BBMV. For both LPD and HPD lowering pH from 7.4 to 6 dramatically increasedK _{0.5 Na} in renal and intestinal BBMV. As compared to pH 7.4, it was enhanced by approximately 200% in both renal and intestinal membranes. This change of Na affinity with acidic pH prevented the expression of P_i transport adaptation at 100 mM Na concentration. However, at saturating Na concentrations (500 mM for renal, 300 mM for intestinal membranes), P_i transport adaptation was equally expressed at pH 6 and 7.4 in both types of membranes. Hill coefficient analysis indicates a 2:1 stoichiometry of Na to P_i in renal and intestinal membranes isolated from high or low P_i diet animals. This ratio was not modified by changes of the medium pH.     

Vitamin D Regulation of Metal loproteinase Activity in Matrix Vesicles

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by l, 25-(OH)₂D₃ [1, 25] and 24, 25-(OH)₂D₃ [24, 25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes were isolated from rat costochondral cartilage and placed into culture. At confluence, GCs were treated with 1, 25 and RCs with 24, 25 for 24 hours. MVs, plasma membranes (PMs), and conditioned media were then collected from the cultures. RT-PCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs. Casein zymography revealed activity at M_r 48 and 28 kDa in MV, but not PM or conditioned media; Western analysis confirmed that this activity was stromelysin-1. Gelatinolytic activity, at low levels, was also found in MVs, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs produced MVs and PMs containing neutral metalloproteinase. Both cells also produced MVs and PMs containing plasminogen activator. The addition of 1, 25 to GCs caused a significant 4- to 5-fold increase in metalloproteinase activity in MVs, but not PMs. In contrast, MVs from cultures of RCs treated with 24, 25 contained decreased metalloproteinase activity; enzyme activity in PMs was unaffected by 24, 25. Plasminogen activator in MVs from RC was increased by treatment with 24, 25, while MV enzyme activity was decreased after treatment of GC cultures with 1, 25. This study shows that both RCs and GCs produce stromelysin-1 and 72 kDa gelatinase and that these enzymes are preferentially localized in MVs. Further, MMP and plasminogen activator activities in MVs and PMs are regulated by vitamin D metabolites.