Biochimica et Biophysica Acta. 1109 (1992) 187-194

1 Q ~¢ • ,:J

© 1992 Elsevier Science Publishers B.V. All rights reserved 0005-273t,/92/$05.00

BBAMEM 75715

The effects of 1,25-dihydroxyvitamin D-3 deficiency on Ca2+-transport and Ca 2+-uptake into brush-border membrane vesicles from pig small intestine Reinhard Kaune, Irina Kassianoff, Bernd Schr6der and Johein Harmeyer Department of PhysiGlogy, School of Veterinary Medicine, Hannm,er (Germany) (Received 30 December 1991) (Revised manuscript received 12 May 1992)

Key words: Brush-hotg~:r membrane vesicle; Calcium ion transport; 1,25.Dihydroxyvitamin D-3; Pseudo vitamin D deficiency; Rickets

BBMV were prepared from duodenal segments of untreated, 1,25-(OH)zDr or vitamin D-3-treated rachitic piglets and from non-rachitic conh'ols by the Mg 2+ precipitation method. The rachitic pigl~t.~ were ,,,,~v,-.~"~'°"";""¢""',,-,...+~"~,.,'Hannover Pig ~,,..,...~,,'~;.",,h;,,h,,...,,.. suffer from pseudo vitamin D-deficiency rickets, type I (no renal l-hydroxylase activity). Initial uptake of Ca2+ (up to 35 s) at low [Ca z+] (between 0.02-0.25 mmol/l) into isolated BBMV consisted of a saturable and non-saturable component. The apparent Vm,,, of the saturable component was significantly lower in rachitie piglets than in coatroi piglets. In the presence of an inside negative potassium diffusion potential, the diffcrcnce in uptake extended over at least 15 rain. The apparent K~ of Ca2+-uptakc was not influenced by the rachitic condition. Treatment of rachitie piglets with sequential doses oi 1,25-(OH)2D 3 for three days (1 ,ag/day) or with a single dose (2.5 rag) of vitamin D-3 elevated the saturable Ca2+-uptake component to values similar to those of control piglets. Addition of 1 mmol/I vcrapamil to the vesicular suspension inhibited Ca2*-uptake in BBMV of control piglets by 40-60% but was without effect on preparations from rachitic piglets. It was concluded from the study that 1,25-(OH)zD 3 dependent Ca2+-uptakc iJ~to BBMV constituted a saturable process which can be inhibited by a know~ Ca2+-channel blocking agent. It appears that 1,25-(OH)2D ~ increases the number of verapamil sensitive Cae+-transport e,miponents in brush-border membranes. The vitamin D-dependent changes in vesicular Ca2+-uptakc were: paralleled by the expression of an active transmural Ca2+-transport across the mucosa.

Introduction The principal effect of 1,25-(OH)zD 3 on intestinal muco~a is stimulation of active Ca2+-transport. This transport is thought to be a three-step process, involving entry of Ca 2+ into the enterocyte across the luminal membrane, its translocation across the cytosol and finally, its active extruzion via the basolaterai membranes [1-31. Hence, at least one nf these steps must to be under control of 1,25-(OH)zD3. Studies of van

Correspondence to: J. Harmeyer, Department of Physiology, School of Veterinary Medicine, Bischofsholer Datum 15, D-300O Hannover 1, Germany. Abbreviations: BBMV, brush-border membrane vesicle(s); 1,25(OH) 2D 3, 1,25-dihydroxTvitamin D-3; 25-(OH)D3, 25-hydroxTvitamin D-3; [Ca 2+ ], total Ca 2+ concentration in medium; AP, activity of alkaline phosphatase; EGTA, etnyleneglycolbis(/]-aminoethyl ether)N,N,N ',N '-tetraacetic acid; Tris, tris(hydroxTmethyl)aminomethane; Hep~s, N.(2.hydroxyethyl)piperaziae-N'-2-ethanesuifonic acid.

Co.wen e~ al. [4] and Kaune et al. [5] with rat and pig duodena! enterocytes have shown that the ATP-dependent transport of Ca 2+ across basolateral membranes was ~:ot it~.flucnced by 1,25-(OH)2D3. The uptake of calcium into isolated brush-border membrane vezicles of chicks and rats, however, has been showq to depend on 1,25-(OH)2D 3. This was concluded from its 30% depression in vitamin-D depleted animals [6-10]. Identification of vitamin D-dependent factors which led to reduced uptake of calcium provided no cleal answers. While a decrease in the K,, of the uptake process and an increase in equilibriura value after 1,25-(OH)2D 3 was reported in some studies [6,7], an increase in Vr~0,,x was obselved in others [8.-10]. In addition, the Ca 2+ concentrations used in these studies we~~ rather high and apparent K m values of about 1 mmo!/l were calculated. No distinction was made b~tween saturable and non-saturable processes. In more recent studies, it has been demonstrated, however, that Ca'-+-uptake into BBMV constituted of a saturable and

188 a non-saturable component [11-14] and that the K~ of the saturable component is much below 1 mmol/l [13,14]. Thus, we reexamined the influence of 1,25(OH)zD 3 upon uptake of Ca :+ into BBMV at low [Ca 2+] (0.02-0.25 mmol/l) and over short incubation times (2-35 s). We used a pig model with inherited rickets. Due to defective renal production of 1,25(OH)2Da [15], these piglets possess un0hysiological low circulating 1,25-(OH)2D3 concentrations [16] and cannot absorb calcium in amounts sufficient to maintain normocaicemia [17]. The animals regularly develop symptoms of florid rickets when they are 5-12 weeks old. Materials and Methods

Anbnals and treatment A number of 25 homozygote piglet~ with type-I pseudo vitamin D-deficiency and symptoms of florid rickets and an equal number of age-matched heterozygote controls of both sexes were used for the experiments [16]. The animals were cross breeds of 'German Landrace' and 'G6ttinger Miniature Pigs' with 3.5-10.5 kg body weight and were 5-12 weeks old. They were weaned at an age of 4-6 weeks and, after weaning, offered a commercial pig starter diet with 0.9% calcium, 0.65% phosphorus and 50/~g vitamin I)-3 per ks feed. For vesicle preparation, the piglets were killed by stunning and bleeding from the carotids. The proximal par~ of the small intestine (duodenum and part of proximal jejunum) was removed, immediately rinsed with ice-cold salire and prepared for incubation of stripped mucosa il, U:~sing-typc chambers. For vesicle preparations the intestine was cut into 15-g pieces and stored at - 70"C. Three rachitic piglets were treated 1 week before tissue preparation with an intramuscular injection of 2,:5 mg vitamin D~ in water sok:~ie fo~m (Sanofi/Ceva, Diisseldorf, Germany).. Three other rachitic piglets were treated three days before tissue preparation with six intravenous injections of 0.5 ~g 1,25-(OH):,D 3 (1 t~g/day) via a catheter placed into the internal jugular vein. The 0.5-~g doses of 1,25-(OH) z D a were dissolved in 50 ~1 ethanol and diluted with 1 ml plasma obtained from the receiver animal. Measurement of C~: + tittles Mucosal to serosal (ms) and serosal to mucosal (sm) fluxes of Ca" ~ were measured in Ussing-type chambers in the ~h~nce of electrochemical gradients by the aid o[ a computer controlled automatic voltage clamp device (AC Microclamp, Aachen, Germany). About 10-cm ~ents from the duodenum were opened along the mesenteric border and fLxed mucosal surface upside on a pyrex plate. Epithelial sheets of tunica mucosa ( 6 - 8

cm 2) were scraped off the underlying muscle layers with a spatula and mounted in Ussing chambers with an exposed area of 1 cm z. To minimize edge damage and to prevent the mucosa from bulging, silicon rubber rings and polyester nettings with open areas > 70% (~,eichelt, Heidelberg, Germany) were placed at both sides of the mucosa. The isolated mucosae were bathed on both sides with 13 ml of buffer solution containing (mmol/i): 137 NaCI, 2.7 KCI, 1.8 CaCi 2, 12 NaHCO 3, 1.0 MgC! 2, 0.4 NaH2PO 4, 0.01 indomethacin and 5.0 Hepes-Tris (pH 7.4). In addition, the serosal solution contained 10 mmc,l/! o-glucose and the mucosal solution 10 mmol/i mannitoi. The ba~hing solutions were continuously circulated and gassed by a ga~ !!ft device with 95% 02 and 5% CO:. The incubation temperature was 39°C. The transepithelial potential difference was continuously measured with 3 mt~I/I KCi agar bridges connected to calomel electrodes. Under short circuited conditions the poter~tial difference was clamped to zero by a computer controlled current via another pair of agar bridges connected to Ag/AgC! electrodes. The tissue conductance was calculated from super-imposed l-s bipolar electrical pulses of 50 tzA amplitude and was recorded along with the potential difference and the short circuit current at 2.5-min intervals. Approx. 30 min after mounting the tissues, 300 kBq 4"~CaCI, was added to one of either side of the mucosa. Samples (0. ! ml) were taken from the labelled side 30 rain later and subsequently in 10 min intervals from the unlabeUed side (0.5 ml) of the Ussing chamber. The samples drawn from the unlabelled side of the mucosa were replaced by equal volumes of isosmotic bathing fluid. Two to three samples were collected under control conditions before 130 pl of verapamd dissolved in incubation medium was added to the lum;: nal surface to give a final concentration of 1 mmol/i. !0-30 rain after addition of verapamil, Ca 2" fluxes were me~,~',~rea.,ar:~ compared to those obtained auring the control period. The difl%rences of ms to sm fluxes were tested between tissues of paired chambers whose conductances did not differ by more than 25%. Net fluxes were e~pressed as differences between ms and sm fluxes ct~tained from paired chambers.

Preparation of BBMV The BBMV were prepared by a modification of the Mg2+-EGTA precipitation method originally described by Biber et al. [18] and Binder and Murer [i9]. Frozen intestinc~ of 20 to 30 g wet weight were thawed and the following procedure wa~; carried out at 4°C. Epithelial cells were harvested after 10 min vibration with a vibro mixer type E 1 (Chemap, M~innedorf, Switzerland) in 3C ml o~. 12 mmol/I Tris.HC! buffer (pH 7.1) with 300 mmol/! mannitol and 5 mmol/I EGTA. Tire sus~ pended cells were diluted 5-fold with 120 ml of ice-cold distilled water and homogenized far 3 min with a

189 blendc: (Muiinex, tyl:,e 530.02, Gc:many). After removal of foam, MgCI: was added to wfinal concentration of 10 mmo b i. The somuun was .~cpt on ice for !5 min and centrifuged at 91}00 × g for 15 min. The supernatant was centrifuged at 27 000 × g for 30 rain and the pellet was resuspended in 35 ml of 5 mmol/! E G T A / T r i s buffer (oH 7.!) with 60 mmol/I mannitol in a glass-teflon-potter (Braun Melsungen, Germany). Precipitation of membranes by Mg 2+ addition and the two-step centrifugation procedure were rr-,,~a!ed. The resulting pellet was :~uspended with a glass-teflon-potter in 35 ml of either a 10 mmol/I Hepes,Fris buffer (pH 7.4) with 100 mmol/! KCI anct 200 mmol/! mannitol or in a 20 mmol/! Hepes-Tris buffer (pH 7.4) with 150 mmol/I KCI and 1 mmol/I MgCI:. This suspension was centrifuged at 270(10 x g for 40 min and the pellet was resuspendcd in 0.5--1.0 ml of the same buffer by passing the mixtr, re several times through a iaypodermic needle (1i.45 ~