The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-atpase activity

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1 & 211 International Society of Nephrology The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-atpase activity Giedre Martuseviciene 1, Jacob Hofman-Bang 1, Torben Clausen 2, Klaus Olgaard 1 and Ewa Lewin 1,3 1 Nephrological Department P, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark; 2 Institute of Physiology, University of Aarhus, Aarhus, Denmark and 3 Nephrological Department B, Herlev Hospital, Copenhagen, Denmark The involvement of sodium/potassium-atpase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the a-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be linked to increased hormone secretion. In this study, we used an in vivo rat model to detere the importance of sodium/potassium-atpase in PTH secretion. Glands were exposed and treated in situ with vehicle or ouabain, a specific inhibitor of sodium/potassium-atpase. PTH secretion was significantly increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by 86 rubidium uptake, which was found to be significantly decreased in ouabain-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also exaed but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium- ATPase activity in vivo had no effect on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model. Kidney International (211) 79, ; doi:1.138/ki.21.1; published online 5 January 211 KEYWORDS: calcium; hyperparathyroidism; ion transport; parathyroid hormone Correspondence: Ewa Lewin, Nephrological Department B, Herlev Hospital, Herlev Ringvej, Herlev DK-273, Denmark. lewin@dadlnet.dk Received 21 September 21; revised 17 October 21; accepted 28 October 21; published online 5 January 211 An enzyme system responsible for active transport across the cell membrane was first identified in 1957 by Skou 1,2 and named Na þ - and K þ -activated ATPase or Na þ,k þ -ATPase. The Na þ,k þ -ATPase or Na þ pump is a protein responsible for the active transport or pumping of Na þ and K þ ions across the plasma membrane, keeping a low concentration of Na þ and a high level of K þ within the cell. The Na þ,k þ -ATPase is expressed in all mammalian cells. It uses energy derived from hydrolysis of adenosine triphosphate to the transport of Na þ and K þ across the plasma membrane with a stoichiometry of 3:2. The Na þ and K þ gradients create the electrical gradient across the plasma membrane, which facilitates cell volume regulation, sodiumcoupled entry of ions, ao acids, glucose, and other compounds, for example, the transcellular transport processes in the intestine, exocrine glands, and kidneys. 3,4 Furthermore, the Na þ,k þ -ATPase might function as a signal transducer. 5,6 Many cell types regulate cytosolic calcium concentration, in part, through a Na þ /Ca 2 þ counter-transporter. In this system, the sodium gradient created by the Na þ,k þ -ATPase is used to transport calcium ions out of the cell. In parathyroid cells, in contrast to most other secretory cells, 7 9 an increase in cytosolic calcium concentration is associated with an inhibition rather than a stimulation of parathyroid hormone (PTH) release. 1,11 Brown et al. 12,13 suggested that the Na þ,k þ -ATPase had a role in the secretion of PTH as they observed that ouabain, a specific inhibitor of the Na þ,k þ -ATPase, and low extracellular potassium inhibited PTH secretion in vitro from bovine parathyroid cells. Dispersed bovine parathyroid cells contained abundant binding sites for ouabain. Inhibition of PTH secretion by ouabain was associated with a concomitant increase in cellular sodium and a reduction in cellular potassium. Brown et al. 13 tested the hypothesis that low K þ and ouabain might modulate PTH release through an increase in cytosolic Ca 2 þ, which was related to alterations in the Na þ Ca 2 þ exchange. However, ouabain and low extracellular potassium caused no significant increase in 742 Kidney International (211) 79,

2 cytosolic Ca 2 þ concentrations in parathyroid cells. In contrast, low K þ caused a decrease in cytosolic Ca 2 þ at high extracellular calcium, whereas ouabain decreased cytosolic Ca 2 þ significantly at all exaed Ca 2 þ concentrations from.5 to 2. mmol/l. 13 Thus, it has remained an open question, how Na þ,k þ - ATPase activity influences parathyroid function. Recently, Imura et al. 14 proposed a novel mechanism linking a-klotho activity and recruitment the of Na þ,k þ -ATPase to the cell surface of the parathyroid cell and thereby to Ca 2 þ -regulated PTH secretion. Klotho is a type 1 membrane protein with similarity to b-glucuronidase. 15 It is predoantly expressed in the kidney, choroid plexus, and parathyroid glands. An important function of klotho in the kidney and possibly also in the parathyroid glands is that of an obligatory co-receptor for fibroblast growth factor ,17 Fibroblast growth factor 23 is a bone-derived hormone that in the kidney suppresses renal phosphate reabsorption and synthesis of calcitriol and in the parathyroids suppresses PTH synthesis and secretion. In addition, an extracellular domain of the klotho protein is shed into the systemic circulation and functions as an endocrine factor. 16,17 Imura et al. observed in vitro in parathyroid tissues obtained from mice that the Na þ,k þ -ATPase was quickly recruited, at low extracellular calcium, to the plasma membrane in connection with the secretion of a-klotho. This pathway was disrupted either by a-klotho deficiency or by adistration of ouabain. There was no significant effect of ouabain on Ca 2 þ -sensitive PTH secretion from a-klotho / glands. Therefore, it was suggested that a-klotho might be required for Na þ,k þ -ATPase-dependent PTH release. 14 Secondary hyperparathyroidism in uremia is characterized by increased PTH-secretory response to hypocalcemia. 18 We have previously shown an increased parathyroid expression of klotho and Na þ,k þ -ATPase in severe hyperparathyroidism, secondary to uremia and hyperphosphatemia in the rat. 19 Therefore, the aim of this investigation was to exae the importance of parathyroid Na þ,k þ - ATPase activity on Ca 2 þ -regulated PTH secretion in vivo in the rat. RESULTS The in vivo effect of ouabain (2 and 1 mmol/l) on low Ca 2 þ -stimulated PTH secretion was exaed. The parathyroid glands were exposed and treated in situ with ouabain, a specific inhibitor of the Na þ,k þ -ATPase (2 mmol/l) or vehicle for 45 before and further for 1 h during the following ethylene glycol tetraacetic acid () infusion. As inbred rats were used, the parathyroid glands had identical size of 1 mm 3 (data not shown). Significant acute hypocalcemia was induced by an intravenous infusion of (Po.1), with no difference in plasma Ca 2 þ between the two groups. As shown in Figure 1a, plasma PTH increased significantly (Po.1) to the same extent in both vehicle- and ouabain-treated rats. Similar results were obtained when the parathyroid glands were treated in situ with 1 mmol/l of ouabain or vehicle for a longer period of time, that is, 12 before and further during the following of infusion, as shown in Figure 1c and d. Significant acute hypocalcemia was induced (Po.1), with no difference in plasma Ca 2 þ between the two groups, and plasma PTH increased significantly (Po.1) to the same extent in both vehicle- and ouabaintreated rats. To ensure that the Na þ,k þ -ATPase pump in the present in vivo model was inhibited by ouabain, the parathyroid glands were exaed for 86 Rubidium ( 86 Rb) uptake. On the basis of the fact that Na þ,k þ -ATPase transports 86 Rb similarly to potassium, 86 Rb uptake by the parathyroid glands was used as an index of Na þ,k þ -ATPase-mediated active sodium potassium transport. The parathyroid glands, exposed in situ to either 1 mmol/l ouabain or vehicle, were removed and incubated separately for 3 in a Krebs - Ringer bicarbonate solution, containing 1. mci/ml 86 Rb. As shown in Figure 2, 86 Rb uptake was significantly decreased in ouabain-treated compared to vehicle-treated parathyroid glands (Po.1), indicating that Na þ,k þ -ATPase activity was inhibited in the ouabain group. As shown in Figure 3, ouabain applied at a high concentration (1 mmol/l) to the parathyroid glands had a clear systemic effect, inducing a significant increase in plasma K þ (Po.1) and a significant decrease in plasma Na þ (Po.1). Therefore, the effect of potassium in vivo on low Ca 2 þ - stimulated PTH secretion was studied by an intravenous infusion of 3 mmol/l KCl as compared with the effect of isotonic NaCl. As shown in Figure 4, plasma K þ levels were significantly increased (Po.1) by KCl infusion, with the high plasma K þ having no significant effect on the PTHsecretory response to low Ca 2 þ. At the time of the peak K þ level (after 3 of KCl infusion), the Ca 2 þ level was slightly lower and the PTH level slightly stimulated before the start of the infusion. However, this difference was not statistically significant. DISCUSSION Exposure of rat parathyroid glands in vivo to high concentrations of ouabain, the specific inhibitor of Na þ,k þ -ATPase activity, had no effect on PTH secretion in this experimental model. No parathyroid cell line is available for the study of PTH secretion. This impedes the possibilities for studying parathyroid cell function. Previous studies describing an effect of ouabain on PTH secretion were all performed in in vitro models of dispersed bovine parathyroid cells or mouse thyroparathyroids ,2 It is well known that parathyroid cells loose their phenotype very fast in in vitro systems Therefore, we decided to use an in vivo model with in situ treatment of the parathyroid glands. A similar model has previously been described by Silver and colleagues. 25 Kidney International (211) 79,

3 Plasma PTH pg/ml Plasma Ca 2+ mmol/l mmol/l Vehicle 6 Plasma Ca 2+ mmol/l Plasma PTH pg/ml mmol/l Vehicle mmol/l mmol/l Figure 1 Effect of ouabain, 2 and 1 mmol/l, on low Ca 2 þ -stimulated PTH secretion in vivo. (a) The parathyroid glands were exposed and treated in situ with ouabain (2 mmol/l), a specific inhibitor of the Na þ,k þ -ATPase, or vehicle for 45 before and for another 6 during an intravenous infusion. (b) Significant acute hypocalcemia was induced by the infusion of (3 mmol/l, at a rate of 3. ml/h; Po.1), with no difference in plasma Ca 2 þ between the two groups. Plasma PTH increased significantly (Po.1) to the same extent in both vehicle- and ouabain-treated rats (panel a). (c) The parathyroid glands were then exposed and treated in situ with ouabain (1 mmol/l) or vehicle for 12 before and for another during the infusion. (d) Significant acute hypocalcemia was induced (Po.1), with no difference in plasma Ca 2 þ between the two groups. Plasma PTH increased significantly (Po.1) to the same extent in both vehicle- and ouabain-treated rats (panel c). Mean±s.e.m., n ¼ 19 in each group., ethylene glycol tetraacetic acid; PTH, parathyroid hormone. 4 4 Vehicle Vehicle We pretreated the parathyroids with ouabain for 2 h before inducing hypocalcemia, and then ouabain treatment was continued for another hour during an infusion. This long-term treatment was based on the observation made by Brown et al. 12 that the in vitro kinetics for the association of ouabain with bovine parathyroid cells was relatively slow and that equilibrium was approached after 2 3 h. Interestingly, Imura et al. 14 performed short-time incubations of 3, and an inhibitory effect of ouabain was seen already after 1 on mouse parathyroids in vitro. In the present in vivo rat model, the stimulating effect of low Ca 2 þ on PTH secretion was neither impaired after a short term (5 ) of ouabain treatment (data not shown) nor after longer time exposure (17 ). Furthermore, no effect of ouabain on PTH secretion was observed after 12 exposure to normocalcemia, despite a significant systemic effect resulting in hyperkalemia and an almost equimolar decrease in plasma sodium. Rothstein et al. 2 used an in vitro model of dispersed bovine parathyroid cells and found that PTH release was inhibited by ouabain at.5 mmol/l calcium, but not at 1. or 3. mmol/l calcium. In the study by Brown et al., 12 an inhibitory effect of ouabain was seen at both.6 and 1. mmol/l calcium, but not at 2. mmol/l. Imura et al. 14 exaed the effect of ouabain only at a low Ca 2 þ concentration of.5 mmol/l. As such, because of these previous in vitro results and the potential impact of inhibition of Na þ,k þ -ATPase activity on Na þ Ca 2 þ exchange in the parathyroid cell, this investigation was designed to focus on a hypocalcemic range of plasma Ca 2 þ concentrations. A continuous infusion of induced a reduction in plasma Ca 2 þ to a very low level of.65 mmol/l. No effect of ouabain was observed in a large spectrum of plasma Ca 2 þ levels, from normal levels of 1.3 to profound hypocalcemia of.65 mmol/l. We used, a calcium chelator, as Brown et al. 12 have shown that did not prevent the inhibitory effect of ouabain on PTH secretion. In a group of rats, in which a high concentration of 1 mmol/l ouabain was applied on the parathyroids, a systemic effect of ouabain was seen with rats developing significant hyperkalemia. As K þ might activate the 744 Kidney International (211) 79,

4 86 Rb uptake CPM/parathyroid gland Plasma K + mmol/l Control 4 Figure 2 Effect of ouabain, 1 mmol/l, on Na þ,k þ -ATPase activity in the parathyroids. 86 Rb uptake by the parathyroid glands was used as an index of Na þ,k þ -ATPase-mediated active Na þ K þ transport. The parathyroid glands were incubated for 3 in Krebs Ringer bicarbonate solution containing 1. mci/ml 86 Rb. The 86 Rb uptake was significantly decreased in ouabaintreated than in vehicle-treated parathyroid glands, Po.1. Mean±s.e.m., n ¼ 18 glands (9 rats) in each group. CPM, counts per ute; 86 Rb, 86 rubidium. Na þ,k þ -ATPase from the extracellular site, 1 a model of hyperkalemic rats was created to exae whether the high levels of K þ by itself would influence PTH secretion. The levels of plasma K þ were comparable with the high levels seen in uremic patients. However, hyperkalemia had no effect on low calcium-stimulated PTH secretion. Brown et al. 26 previously exaed the effect of potassium on PTH secretion from bovine parathyroid cells. They found that removal of extracellular potassium markedly inhibited PTH secretion at low calcium in a dose-related manner, whereas no effect of increasing potassium above 2 3 mmol/l was seen. 26 In another study, Shobac and Brown 27 compared the effect of 5 6 mmol/l potassium concentration and found that PTH release was stimulated by 6 mmol/l K þ. Such an extreme extracellular potassium concentration is of no relevance for an in vivo model. However, potassium ions were shown to reduce the efficacy of the Na þ,k þ -ATPase for ouabain, predoantly through a reduction in the association rate. 28 Therefore, in the present model, the effective inhibition of Na þ,k þ -ATPase activity in the parathyroids was tested by measuring 86 Rb uptake by the parathyroid glands Rb uptake was significantly inhibited by ouabain exposure in this in vivo model. The concentrations of ouabain used in the present investigation and in the paper by Imura et al. 14 were very high. Imura et al. observed an effect of 1 mmol/l ouabain on mice parathyroids in an in vitro model. In the present investigation, we used a concentration of 2 mmol/l of topically applied ouabain for exposure to the parathyroids, as a local external application potentially is less effective, than exposure through the medium to cells in vitro. Plasma Na + mmol/l Control Figure 3 Systemic effects of ouabain. applied in vivo at a high dose of 1 mmol/l to the parathyroid glands had a clear systemic effect, inducing a significant increase in plasma K þ (Po.1) and a significant decrease in plasma Na þ, Po.1. Mean±s.e.m., n ¼ 9 in each group. As no effect of ouabain was observed at a concentration of 2 mmol/l, a much higher concentration of 1 mmol/l was used. Still, no effect on the PTH-secretory response to hypocalcemia was observed, despite the fact that a clear systemic effect of ouabain was obtained. The Na þ,k þ -ATPase is a large protein complex. The imum functional unit is a heterodimer of a- and b-subunits. Individual genes of four a-subunit isoforms and at least three b-subunit isoforms of the Na þ,k þ -ATPase have been identified in mammals. The isoforms combine to form a number of Na þ,k þ -ATPase isozymes that are expressed in a tissue- and cell-specific manner. The specific regulation of the expression of these isozymes in rat parathyroids is out of the scope of this investigation. In most mammalian species, the Na þ,k þ -ATPase, irrespective of isoform composition, has a very high affinity to the specific inhibitor, ouabain. This is Kidney International (211) 79,

5 true except for some rodent species, mainly the rat, in which pumps containing the a1-isoform have a relatively low affinity for cardiac glycosides. 3 Plasma K + mmol/l Plasma PTH pg/ml Plasma Ca 2+ mmol/l However, the important observation is that in the present investigation, in an in vivo model, no effect of ouabain was observed on the PTH-secretory response to hypocalcemia, despite significant inhibition of the pump activity, as shown by the inhibited 86 Rb uptake by the parathyroid glands, when ouabain was applied. We have previously found an increased parathyroid expression of the Na þ,k þ -ATPase together with an increased expression of klotho in severe hyperparathyroidism, secondary to uremia and hyperphosphatemia in the rat. 19 Therefore this investigation was conducted with the expectation that the Na þ,k þ -ATPase might be of relevance for enhanced PTH secretion in uremia. However, the present results do not support such a mechanism. Conclusion Inhibition of the parathyroid gland Na þ,k þ -ATPase activity had no effect in vivo on the PTH-secretory response to acute hypocalcemia in the rat. Thus, the previously suggested importance of the Na þ,k þ -ATPase in the regulation of PTH secretion could not be confirmed in the present in vivo rat model. MATERIALS AND METHODS Animals The experimental studies were performed in accordance with the Danish law and approved by the Ministry of Justice. Inbred male dark auguti rats, weighing 2 g (Taconic A/S, Ejby, Denmark) were used. Rats were fed a standard diet (.9% calcium,.7% phosphorus, and vita D, 1 IU/kg), with free access to food and water. They were kept in a controlled environment in a 12-h light dark cycle, constant temperature (of 22 1C), and relative humidity (7%). The total number of rats amounted to 92. Rats received anesthesia with pentobarbital ( mg/kg; Nycomed-DAK, Copenhagen, Denmark), intraperitoneally. Additional doses were adistered, when required, to maintain a steady level of anesthesia. Induction of hypocalcemia by infusion of Hypocalcemia was induced by intravenous infusion of (3 mmol/l; Sigma-Aldrich, St Louis, MO), at a rate of 3. ml/h for 6 through a catheter inserted into the femoral vein. The method has been described elsewhere Samples for deteration of plasma PTH, Ca 2 þ, Na þ, and K þ concentrations were obtained from a corresponding catheter in the femoral artery at, 5, and every 1. Each time a blood sample was taken, the blood volume was replaced by an intravenous adistration of.4 ml saline. Figure 4 Effect of potassium on low Ca 2 þ -stimulated PTH secretion in vivo. As the application of ouabain on the parathyroids increased plasma K þ, the effect of potassium on low Ca 2 þ -stimulated PTH secretion in vivo was studied by an intravenous infusion of 3 mmol/l KCl as compared with the effect of isotonic NaCl adistered for 3, followed by an infusion. Plasma K þ levels were significantly increased (Po.1) by the KCl infusion. An increase in plasma K þ had no effect on the PTH-secretory response to low Ca 2 þ. Mean ± s.e.m., n ¼ 8 in each group., ethylene glycol tetraacetic acid; PTH, parathyroid hormone. 746 Kidney International (211) 79,

6 Application of ouabain The parathyroid glands were exposed and treated in situ 25 with ouabain (C 29 H 44 O 12-8 H 2 O; Sigma-Aldrich, St Louis, MO), a specific inhibitor of the Na þ,k þ -ATPase or vehicle. All procedures were performed by microsurgery using a stereomicroscope. A microscopic nest of cotton was built and applied over the parathyroid gland. A volume of 1 ml ouabain or vehicle was then placed over the nest and renewed every Rb uptake The uptake of 86 Rb (Perkin-Elmer Life and Analytical Sciences, Boston, MA) by the parathyroid glands and the inhibition of the uptake by ouabain were used as a measure of Na þ,k þ -ATPasemediated active sodium potassium transport. Two parathyroid glands from either ouabain-exposed rats or vehicle-exposed control rats were incubated separately for 3 at 3 1C in 2 ml of Krebs Ringer bicarbonate solution (Sigma- Aldrich) in Corning Costar Transwell Polycarbonate Inserts (Fisher Scientific, Slangerup, Denmark), containing 1. mci/ml 86 Rb. To remove 86 Rb from the extracellular space, the glands were then washed 4 15 in 1 ml of an ice-cold sodium-free Tris (1 mmol/l)-sucrose (2 mmol/l) buffer (from Sigma-Aldrich). After washing, the glands were transferred to counting vials, containing 2 ml 5% trichloroacetic acid, and then radioactivity was measured in a b-counter after adding 2.5 ml Ultima Gold (Perkin-Elmer Life and Analytical Sciences, Waltham, MA). 29 As the parathyroid glands of inbred DA rats had identical size, the results are presented as c.p.m. (counts per ute) per gland. Experimental protocols Effect of ouabain, 2 mmol/l, on low Ca 2 þ -stimulated PTH secretion in vivo. The parathyroid glands were exposed and treated in situ 25 with ouabain (2 mmol/l) or vehicle for 45 before and for another 6, during induction of hypocalcemia by an infusion (n ¼ 19 in each group). Effect of ouabain, 1 mmol/l, on low Ca 2 þ -stimulated PTH secretion in vivo. The parathyroid glands were exposed and treated in situ with ouabain (1 mmol/l) or vehicle (n ¼ 19 in each group) for 12 before and for another during infusion. The parathyroids were then removed and inhibition of the Na þ,k þ -ATPase was exaed by 86 Rb uptake (n ¼ 9). Effect of potassium on low Ca 2 þ -stimulated PTH secretion in vivo. Intravenous infusion of 3 mmol/l KCl or isotonic NaCl (3 ml/h) was performed for 3, followed by an infusion (n ¼ 8). Samples for plasma PTH, Ca 2 þ, Na þ, and K þ were obtained every 1. Plasma deterations Plasma phosphate, urea, and creatinine were analyzed using Vitros 2 (Ortho-Clinical Diagnostics, Rochester, NY). Plasma Ca 2 þ,na þ,and K þ were measured by ABL 5 (Radiometer, Denmark). Plasma PTH was measured by a rat-bioactive intact PTH ELISA (enzyme-linked immunosorbent assay) (Immunotopics, San Clemente, CA). 31 Statistics Student s t-test or Mann Whitney test and ANOVA (analysis of variance)withbonferroni sposttestwereusedanddataarepresented as mean±s.e.m. Po.5 was considered statistically significant. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS We are very grateful to Kirsten Bang and Ulla Steen Petersen, both technicians, for their wonderful collaboration and very skillful work. We thank The Foundation of and the Simon Fougner Hartmanns Family Foundation for their financial support. REFERENCES 1. Skou JC. Nobel lecture. The identification of the sodium pump. Biosci Rep 1998; 18: Skou JC. The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim Biophys Acta 1957; 23: Jorgensen PL, Hakansson KO, Karlish SJ. Structure and mechanism of Na,K-ATPase: functional sites and their interactions. 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