The involvement of intracellular calcium ion concentration and calmodulin in the 25-hydroxylation of cholecalciferol in ovine and rat liver.

The effect of Ca2+ ion concentration on the 25 hydroxylation of tritiated cholecalciferol (3HD3) was investigated using homogenates of ovine liver from vitamin D replete sheep. A significant decrease in the production of 25 hydroxycholecalciferol (25OHD3) was observed when the concentration of Ca2+ in the homogenate was raised above 0.68 mmol/l by the addition of calcium gluconate. Similarly, a final concentration of 37 mumol EGTA/1 (equivalent to a Ca2+ concentration of 26.5 nmol/l) was associated with a 50% reduction of 25OHD3 production. That is, a broad bell-shaped relationship was observed between the production of 25OHD3 and the Ca2+ concentration in the homogenate. These changes in the rate of production of 25OHD3 were reproduced with hepatocytes from vitamin D replete rats, prepared by collagenase perfusion, using the drugs dantrolene sodium (DaNa) to reduce (ED50 = 57 mmol/l) and veratridine to increase (ED50 = 550 mmol/l) the intracellular Ca2+ concentration. Hepatocytes from vitamin D replete rats also showed a reduction in 25 hydroxylation of D3 (ED50 = 6 ng/ml) in response to the addition of 1-25 dihydroxycholecalciferol (1-25 (OH)2D3). The calmodulin antagonists; W7, compound 48/80, trifluoperazine (TFP) and calmidazolium (R24571) were all found to effect a dose response inhibition of the 25 hydroxylation of cholecalciferol by homogenates of ovine liver. R24571 had a similar inhibitory effect (ED50 = 70 mumol/l) upon the 25 hydroxylase enzyme of rat hepatocytes. It is concluded that the 25 hydroxylation of cholecalciferol in liver of vitamin D replete rats and sheep is calcium sensitive and is reduced in the presence of increased concentrations of 1,25(OH)2D3. Calmodulin may also be involved in the regulation of hepatocyte 25-hydroxylase activity by Ca2+.

Effect of low calcium and low phosphorus diets on the intestinal absorption of phosphate in intact and parathyroidectomized pigs.

The intestinal absorption of phosphate has been studied in conscious pigs, each prepared with a Thiry-Vella loop of jejunum. The feeding of diets low in either calcium or phosphorus caused a significant increase in the efficiency of absorption of phosphate from the solution used to perfuse the jejunal loop in both intact and parathyroidectomized (PTX) pigs. An intravenous infusion of parathyroid hormone (0.22 u. kg-1 h-1) into a PTX pig also enhanced the absorption of phosphate. The increase in the absorption of phosphate when the low phosphorus diet was fed was not caused by an increase in the concentration gradient of phosphate ions between the jejunal lumen and blood. It is concluded that the intestinal absorption of phosphate shows similar changes to those of calcium when diets low in calcium or phosphorus are fed and that parathyroid hormone, although capable of stimulating the absorption of phosphate, is not essential for this adaptation. These effects are probably brought about by changes in the renal production and mucosal uptake of 1,25-dihydroxycholecalciferol, the active metabolite of vitamin D3.

Reversal of betamethasone-induced inhibition of intestinal calcium absorption by 1alpha-hydroxycholecalciferol.

The intestinal absorption of calcium has been studied in conscious, unstressed pigs, using a modification of the double isotope technique. The oral administration of betamethasone (1 mg/day) to four pigs (25--33 kg) for 4 weeks reduced the calcium absorption coefficient, calculated after the intravenous and oral administration of 47Ca2+, by a mean value of 66%. The oral administration of 1alpha-hydroxycholecalciferol (2 microgram/day) in combination with beta-methasone (1 mg/day) for a further 4 weeks returned the absorption coefficient to the control value.

Effects of low calcium and low phosphorus diets on the duodenal absorption of calcium in betamethasone-treated chicks.

The effect of oral administration of betamethasone (25 microgram kg-1 day-1) on the duodenal absorption of calcium has been studied in chicks using the ligated loop technique in vivo. The chicks were fed normal calcium, normal phosphorus (NCaNP), low calcium, normal phosphorus (LCaNP) or normal calcium, low phosphorus (NCaLP) diets. Daily oral administration of betamethasone for 2-3 weeks markedly reduced the absorption of calcium in chicks fed the NCaNP diet, but did not significantly affect the adaptation in absorption when the NCaLP or LCaNP diets were fed for the same period of time. In one group of chicks, betamethasone was administered daily for 10 days before the birds were transferred to the NCaLP or LCaNP diets. Adaptation was again unaffected by betamethasone treatment. Administration of betamethasone caused a marked retardation in growth-rate, hypercalcaemia and an increased percentage of ash in the tibiae.

Effect of 1,25-dihydroxyvitamin D3 on plasma concentrations of calcium-binding protein in normal and rachitic (vitamin D-dependent rickets type I) pigs.

An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2.5 h on plasma concentrations of calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4-8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4-8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1.5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels. In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.

Plasma vitamin D-binding protein and free 1,25-dihydroxyvitamin D3 index in pregnant ewes and their fetuses in the last month of gestation.

A radioimmunoassay for ovine vitamin D-binding protein (DBP) has been developed. This assay can also effectively measure DBP in goat plasma. A suitable ovine DBP antiserum raised in a rabbit produced a single monospecific line of precipitation when reacted against purified sheep DBP and sheep plasma. The preliminary purification of 125I-labelled ovine DBP was carried out using adsorption chromatography, and the final purification immediately before addition to the assay tubes was achieved by high-pressure liquid chromatography. Displacement of 125I-labelled ovine DBP by dilutions of sheep and goat plasma or standard DBP gave parallel curves, and only weak competition was observed with calf and pig plasma. The assay detected as little as 26 pmol DBP/l with intra- and interassay coefficients of variation of 3 and 14% respectively. The mean plasma concentration of DBP in nine pregnant sheep (110-120 days of gestation) was 8.7 +/- 0.3 (S.E.M.) mumol/l. These levels were significantly (P less than 0.02; paired t-test) higher than those in matched fetal plasma (6.7 +/- 0.4 mumol/l) obtained in utero through a catheter in a carotid artery. Plasma DBP concentrations in pregnant sheep were also significantly (P less than 0.02) higher than in five normal non-pregnant sheep (6.8 +/- 0.5 mumol/l). The mean concentrations of total 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in maternal and fetal plasma were 92.0 +/- 8.7 pmol/l and 152.5 +/- 18.0 pmol/l respectively (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine control of plasma concentrations of calcium-binding protein in the pig.

The aetiology of the rise in plasma calbindin-D9K (vitamin D-induced calcium-binding protein; CaBP), following insulin-induced hypoglycaemia, was studied in the pig. ACTH led to a rise in plasma concentrations of both CaBP and cortisol. Metyrapone, which blocks cortisol synthesis, abolished the increases in plasma concentrations of CaBP and cortisol normally observed in response to insulin-induced hypoglycaemia. However, there was no significant rise in plasma concentrations of CaBP in response to pharmacological or physiological doses of cortisol. Injection of clonidine, an alpha 2-adrenergic agonist, led to a rise in plasma concentrations of CaBP, whereas phenylephrine, an alpha 1-adrenergic agonist, tended to exert an inhibitory effect. Also, administration of phentolamine (an alpha-adrenergic blocker) before injection of insulin abolished the usual increase in plasma concentrations of CaBP, whereas propranolol (a beta-adrenergic blocker) enhanced the normal increase in plasma concentrations of CaBP in response to insulin-induced hypoglycaemia. Isoproterenol, a beta-adrenergic agonist, was without effect on plasma CaBP. Neither GH nor glucagon appear to be involved in the rise in plasma CaBP following insulin-induced hypoglycaemia. Although atropine abolished the effect of acute hypoglycaemia on plasma CaBP, carbamylcholine was without effect on plasma CaBP concentration. It is concluded that the increases in plasma CaBP induced by either ACTH or alpha 2-adrenergic stimulation may be interrelated since the administration of ACTH can lead to raised plasma concentrations of catecholamines.

Production of parathyroid hormone-related protein by the mammary gland of the goat.

Parathyroid hormone-related protein (PTHRP) has been quantified by sensitive specific immunoassays in mammary venous blood and milk from 7 days before to 7 days after parturition in the goat. A significant venous-arterial concentration gradient in plasma PTHRP 1-86 concentrations was demonstrated across the mammary gland, indicating that PTHRP enters the maternal circulation and may have a role in calcium homoeostasis during lactation. Significant and sustained increases in mammary venous and milk PTHRP 1-86 concentrations were found from 1 day before parturition to 7 days afterwards, with peak concentrations of 1.57 +/- 0.58 pmol/l (plasma) and 8.69 +/- 2.95 nmol/l (milk) (mean +/- S.E.M.) occurring on day -1 and the day of parturition respectively. Estimates of the mammary output of PTHRP into plasma in four goats averaged 9% (range 1-25%) of that secreted into milk. Suppression of maternal prolactin concentrations by bromocriptine significantly reduced milk yield and the mammary venous PTHRP concentration, without affecting the concentration of PTHRP in milk. In conclusion, parturition in the goat is associated with a sustained increase in secretion of PTHRP into both plasma and milk; the former may be involved in maternal calcium homoeostasis, whereas the latter may have a role in the neonate.

Effect of low phosphorus diets on intestinal calcium absorption and the concentration of calcium-binding protein in intact and parathyroidectomized pigs.

The effect of changing the dietary concentration of phosphorus on the intestinal absorption of calcium has been studied in conscious pigs each prepared with a Thiry--Vella loop of jejunum. A reduction in the percentage of phosphorus in the diet from 0.7 to 0.3% caused an increase in the efficiency of absorption of calcium from the fluid used to perfuse the jejunal loop in both intact and parathyroidectomized animals. There was a marked increase in the amount of calcium-binding protein (CaBP) in the small intestine of pigs fed the low phosphrous diet. Parathyroidectomy did not affect the amount of CaBP in the small intestine when either the normal or the low phosphorus diets were fed.

Stimulation by low phosphorus and low calcium diets of duodenal absorption of phosphate in betamethasone-treated chicks.

Although the inhibitory effect of glucocorticoids on the intestinal absorption of calcium is well recognized, their effect on the absorption of phosphate is less well documented. We studied the effect of the oral administration of betamethasone (BM; 25 micrograms/kg per day) on the duodenal absorption of phosphate in chicks fed normal calcium, normal phosphorus (NCaNP), normal calcium, low phosphorus (NCaLP) or low calcium, normal phosphorus (LCaNP) diets using the ligated loop technique in vivo. The daily oral administration of BM for 8 days significantly reduced the absorption of phosphate in chicks fed the NCaNP diet (21% decrease) but had less effect in chicks fed the NCaLP (14% decrease) or LCaNP (9% decrease) diets in which birds the absorption of phosphate was significantly raised (49 and 87% respectively). In one group of chicks, BM was administered for 9 days before the birds were transferred to the NCaLP or LCaNP diets. Adaptation was again unaffected by the treatment. Thirty-four per cent of the absorbed phosphate was retained in the duodenal tissue. Treatment with BM reduced the amount retained but this may have been caused by the lower weight of the duodenal segment in these chicks as BM administration markedly reduced growth rate. We have concluded that the duodenal absorption of phosphate in the chick can be inhibited by treatment with BM, although this may be secondary to the reduced rate of growth, but the increase in the absorption of phosphate caused by feeding NCaLP or LCaNP diets was unaffected by the steroid.

Raised levels of calcium-binding protein in plasma following insulin-induced hypoglycaemia in the pig.

Insulin-induced hypoglycaemia in the pig elicited sharp increases in the plasma concentrations of vitamin D-dependent calcium-binding protein (CaBP) and cortisol and a decrease in plasma inorganic phosphate. Glucose infusion following insulin administration abolished the increases in plasma CaBP and cortisol in response to insulin and reduced the hypophosphataemia. The percentage increases in plasma CaBP and cortisol in response to insulin-induced hypoglycaemia were reduced when the pigs were fed a low-calcium diet, but the hypophosphataemic response was similar. We conclude that insulin-induced hypoglycaemia leads to increased plasma CaBP in pigs fed a normal calcium diet, which is associated with the hypoglycaemia rather than being a direct effect of insulin. We therefore suggest that plasma CaBP may represent more than a mere uncontrolled leak from its sites of storage.

Clearance of exogenous parathyroid hormone-related protein in pregnant, non-pregnant and fetal sheep, goats and pigs.

In the sheep, goat and pig, radiolabelled parathyroid hormone related protein (PTHRP) and immunoreactive PTHRP(1-34) and (1-86) were rapidly cleared from the circulation. Metabolic clearance rates (MCR) were in the range of 1.25-7.5 ml/min per kg and were slightly slower than that of intact PTH in man (10 ml/min per kg); while the mean MCR of labelled PTHRP(1-86) in fetal sheep and goats was significantly faster than that in their respective mothers (14.4 vs 4.0 ml/min per kg respectively). This may reflect increased metabolism of PTHRP by fetal tissues, e.g. the placenta. Similar rates of clearance of radiolabelled PTHRP(1-141), (1-86) and (1-34) suggest that clearance involves the amino terminus of the molecule.

Evidence for a novel parathyroid hormone-related protein in fetal lamb parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcaemia of malignancy.

Parathyroid hormone (PTH)-like bioactivity, assayed as adenylate cyclase response in UMR 106-01 osteogenic sarcoma cells, was present in extracts of sheep fetal and maternal parathyroid glands and placenta. Preincubation of extracts with PTH(1-34) antiserum inhibited approximately 40% of the bioactivity in fetal parathyroid extracts, 50% in maternal parathyroid extracts, but only 10% of the bioactivity in the placental extract. Partial purification of placental extracts by chromatography yielded fractions containing PTH-like bioactivity which were similar in behaviour to that of PTH-related protein (PTHrP) from a human lung cancer cell line (BEN). An antiserum against synthetic PTHrP(1-16) partially inhibited the bioactivity of the placental extract and synthetic PTHrP(1-34), but had no effect on the bioactivity of bovine PTH(1-34) or bovine PTH(1-84). The placental PTH-like bioactivity was higher in mid- than in late gestation. Fetal parathyroid glands contained the highest PTH-like bioactivity. Thyroparathyroidectomy of one fetal twin lamb in each of 16 ewes between 110 and 125 days of gestation resulted in decreases of the plasma calcium concentration and reversal of the placental calcium gradient that existed between the ewe and the intact fetus. Perfusion of the placenta of each twin in anaesthetized ewes was carried out sequentially with autologous fetal blood in the absence of the exsanguinated fetus. The plasma calcium concentration in the blood perfusing the placenta of each twin increased, but reached a plateau at a lower concentration in the perfusing blood of thyroparathyroidectomized fetuses than in that of the intact fetuses. Addition of extracts of fetal parathyroid glands or of partially purified PTHrP resulted in further increases in plasma calcium in the autologous blood perfusing the placentae of thyroparathyroidectomized fetuses, but addition of bovine PTH(1-84) or rat PTH(1-34) had no effect. The presence of this PTH-like protein in the fetal parathyroid gland and placenta may contribute to the relative hypercalcaemia of the fetal lamb. This protein, which is similar to PTHrP associated with humoral hypercalcaemia of malignancy, stimulates the placental calcium pump responsible for maintaining a relative fetal hypercalcaemia during gestation.

The transfer of calcium during perfusion of the placenta and intact and thyroparathyroidectomized sheep.

Placental perfusions were carried out in six ewes during the last two weeks of gestation. Perfusions were carried out using autologous fetal blood and the flow rates adjusted to give a perfusion pressure of 50--70 mmHg. Perfusion plasma calcium concentrations rose steadily throughout the perfusions achieving a mean increase of 1.65 mmol/1 above initial concentration within 100 minutes. A further three ewes in the last two weeks of gestation were thyroparathyroidectomized and normal plasma calcium concentration maintained by an intravenous infusion of calcium borogluconate. After three days, placental perfusions were carried out as before. The mean perfusion plasma calcium concentration achieved by those three ewes in a period of 100 minutes showed an increase of 1.25 mmol/1. It is concluded that the presence of the fetus is not necessary for the continued active transfer of calcium across the placenta from mother to fetus. The reduced rate of accumulation of calcium in the perfusate in TXPTX ewes is attributed to a decline in 1,25-DHCC concentrations in both maternal and fetal circulations. The implications of these results in relation to fetal calcium homeostasis and the placental transfer of calcium are discussed.

Genetic regulation of placental function: a quantitative in situ hybridization study of calcium binding protein (calbindin-D9k) and calcium ATPase mRNAs in sheep placenta.

The calcium requirement of the ovine fetus increases progressively throughout pregnancy. The 9-kDa calcium binding protein (calbindin-D9k; 9CBP) is considered to be a reliable marker for epithelia mediating calcium transport. This quantitative in situ hybridization study shows that the levels of 9CBP mRNA show a pregnancy stage-related increase which correlates with fetal calcium demand only in maternal endometrial gland and fetal interplacentomal trophoblast epithelia. Levels of 9CBP mRNA in the placentome, which has by far the greater area of maternofetal contact, show no changes during pregnancy. mRNA for the CaATPase enzyme, a second requirement for calcium transport, is shown to be present in epithelia in interplacentomal and placentomal regions but shows no change in concentration as pregnancy progresses. Results with the 9CBP and CaATPase mRNAs confirm our recent immunocytochemical results with ruminant placenta and indicate the basis for a cellular calcium transport system analogous to that in the enterocyte. The interplacentomal trophoblast system appears to be eminently suitable for investigations of details of the cellular mechanism and control of epithelial calcium transport.

Aspects of calcium transport by the ovine placenta: studies based on the interplacentomal region of the chorion.

An in vitro technique for the measurement of calcium uptake into the maternal-facing fetal chorionic membrane (apical trophoblast) was used to study the relationship between calcium uptake and stage of pregnancy in the sheep. The effects on calcium uptake of varying calcium concentration and temperature of the incubation medium, of adding calcium channel blockers or heavy metals (lanthanum and nickel) or calcium ionophore/agonist were also studied. The data indicate a saturable calcium uptake process, plateauing after 15 min incubation. This uptake remained constant throughout the last third of gestation until a significant fall in uptake was noted during the final week prior to parturition. This uptake was not due to extracellular cellular diffusion since there was no significant uptake of tritiated inulin over the same period in each case. Calcium uptake in this system was also shown to be a temperature dependent process which was abolished at temperatures of 0-4 degrees C. A decrease in calcium concentration to 0.12 mM in the incubation medium also caused a corresponding decrease in calcium uptake to 21 per cent of control (1.2 mM). The addition of the heavy metals lanthanum and nickel also significantly reduced calcium uptake as did the calcium channel blockers verapamil, metoprolol and diltiazem. The calcium channel ionophore A23187 increased calcium uptake into the material facing chorion. Although the interplacentomal chorion may not be representative of the whole of the placental unit, it clearly contains a specific calcium uptake process under local physiological control. The blocking of calcium uptake by the specific I-type calcium channel blocker verapamil may indicate the presence of I-type channels of unusually low sensitivity since the concentration needed to block them was much higher than would be required for excitable I-type channels in isolated cells.

The effect of fetal thyroparathyroidectomy on the transport of calcium across the ovine placenta to the fetus.

The ovine fetal placenta has been perfused with autologous fetal blood under controlled conditions in eleven experiments in which the fetus was first removed. Eight of these experiments involved four pairs of twins, one lamb of which had been thyroparathyroidectomized (TXPTX) three to seven days earlier. By this time the normal placental calcium gradient from mother to fetus had either decreased or been reversed. The mean rate of transport of calcium from the mother was unchanged by previous fetal TXPTX, but the final calcium gradient achieved from the mother to the perfusing blood was significantly less than with placentae from intact fetuses. No significant alteration in fetal plasma I,25-dihydroxyvitamin D (I,25(OH)2D) concentration was observed as a result of the fetal TXPTX, indicating that hypocalcaemia can compensate for the lack of PTH in fetal production of I,25(OH)2D. Fetal thyroidectomy with replacement of thyroxine did not lead to reversal of the placental calcium gradient, indicating that calcitonin was not involved. It is suggested that in the ovine fetus, parathyroid hormone promotes the active transport of calcium from mother to fetus, so that in its absence the fetus must obtain its calcium for growth by reducing its calcaemia and thereby allow net diffusion of calcium to replace the action of the placental calcium pump. The price paid for this compensation is marked hypocalcaemia and defective calcification of osteoid.

Role of the fetal parathyroid glands and parathyroid hormone-related protein in the regulation of placental transport of calcium, magnesium and inorganic phosphate.

The plasma Ca concentration of the fetus is maintained higher than maternal levels by active placental transport. Ca, Mg and PO4 accumulation by the fetus is mainly associated with skeletal growth. The fetal parathyroid glands are essential for maintenance of elevated plasma Ca, which is necessary for the stimulation of fetal osteoblasts and mineralization of cartilage and osteoid. Fetal thyroparathyroidectomy (TxPTx) results in a decreased activity of the placental Ca pump. The presence of a parathyroid hormone-related protein (PTHrP) has been demonstrated in fetal parathyroid glands and placental tissue. Extracts of fetal parathyroid glands and purified PTHrP, as well as recombinant PTHrP (1-84, 1-108 and 1-141), stimulate Ca and Mg but not PO4 transport across the placenta of TxPTx-ized fetuses perfused with autologous blood in the absence of the fetus. Parathyroid hormone (PTH) and the N-terminal region of PTHrP do not stimulate placental Ca and Mg transport. It is concluded that a mid-molecule region of this novel hormone may be required to stimulate placental Ca transfer and contribute to the regulation of fetal Ca homeostasis.

Relationship between intrafollicular concentrations of parathyroid hormone-related peptide (PTHrP) and steroid hormones in oestrogenic and non-oestrogenic ovarian follicles in the mare.

The objective of the present study was to determine whether parathyroid hormone-related peptide (PTHrP) is present in the equine follicular fluid and if so, how it is related to the follicular development in the horse. For this purpose, ovaries were collected from 40 Thoroughbred and Thoroughbred Cross mares at slaughter during the period from February to May. Normal growing follicles were dissected from the ovaries of each mare and their diameters measured. A total of 174 follicles was used in this study. The follicular fluid was aspirated from each follicle and assayed for PTHrP, oestradiol (E), testosterone (T) and progesterone (P). The follicles were classified as either oestrogenic or non-oestrogenic if the follicular fluid content of oestradiol was >40 or <40 ng/ml, respectively. PTHrP concentrations were significantly (P<0.05) higher in oestrogenic follicles, but T and P concentrations did not differ. Furthermore, E:T ratio was significantly (P<0.05) greater in oestrogenic follicles compared to the non-oestrogenic ones. The mean diameter of oestrogenic follicles was significantly (P<0.05) greater than that of non-oestrogenic ones. The higher concentrations of PTHrP observed in the follicular fluid of healthy oestrogenic follicles suggest that it may have a role in the control of ovarian function.

Lack of effect of quinidine on divalent mineral absorption from the reticulorumen of sheep.

The effects of quinidine on divalent mineral absorption were studied using normal and high potassium intraruminal buffers with the in vivo isolated washed rumen technique. A concentration of 1 mmol litre-1 quinidine in the intraruminal buffer decreased the absorption rates of Na+ and C1- from the rumen but had no significant effect on the absorption rates of Mg2+, Ca2+ and K+. Prolonged oral administration of quinidine had no significant effect on the absorption of any of these ions from either a normal or a high potassium ruminal solution.