Regulation of parathyroid hypertensive factor secretion by vitamin D3 analogs in parathyroid cells derived from spontaneously hypertensive rats

AJH–May 2003–VOL. 16, NO. 5, PART 2

P-362 ELISA DETECTION OF PARATHYROID HYPERTENSIVE FACTOR IN NORMOTENSIVE AND HYPERTENSIVE INDIVIDUALS Svetlana Krylova, Teresa Labedz, Richard Z Lewanczuk, Christina G Benishin. Research, CV Technologies, Edmonton, Alberta, Canada; Physiology, University of Alberta, Edmonton, Alberta, Canada; Endocrinology, University of Alberta, Edmonton, Alberta, Canada. Preliminary human studies suggest that parathyroid hypertensive factor (PHF) may be a useful marker in the detection and treatment of essential hypertension. In particular, PHF positive patients have been shown to respond most favorably to treatment with calcium channel blockers. Anti-PHF monoclonal antibodies were developed using standard procedures, and used to screen plasma samples from normotensive and hypertensive (untreated) patients for PHF levels. The anti-PHF antibodies were capable of inactivating PHF biological activity (blood pressure bioassay) ex vivo. There was no detectable cross-reactivity with human or rat parathyroid hormone (PTH). PHF levels measured with ELISA were correlated with bioassay measurement of PHF levels in the same samples. The monoclonal antibodies reacted equally with rat and human PHF. In normotensive patients, the average level of circulating PHF as measured with monoclonal anti-PHF ELISA is 0.1819 ⫾ 0.0207 Units/ml (N⫽49), and was not correlated with mean arterial pressure (r⫽0.0488, not significant). In hypertensive patients (mean arterial pressure ⱖ 95 mm Hg) who were not on any medications (N⫽27), circulating PHF levels were elevated up to three fold, and positively correlated with mean arterial pressure (r⫽0.394, significant). These results support previous studies describing the correlation between PHF and hypertension and indicate that the selective monoclonal antibodies may provide an improved ELISA method for clinical assessment of hypertension. Furthermore, the detection of significant levels of PHF in normotensive individuals as well as in hypertensives could suggest that PHF has a physiological function and may be classified as a hormone. Key Words: parathyroid hypertensive factor, calcium, monoclonal antibody

P-363 REGULATION OF PARATHYROID HYPERTENSIVE FACTOR SECRETION BY VITAMIN D3 ANALOGS IN PARATHYROID CELLS DERIVED FROM SPONTANEOUSLY HYPERTENSIVE RATS Sharla K Sutherland, Ilka Nemere, Christina G Benishin. Physiology, University of Alberta, Edmonton, Alberta, Canada; Nutrition and Food Sciences, Utah State University, Logan, UT. Parathyroid Hypertensive Factor (PHF) is a novel substance secreted by the parathyroid gland (PTG), and which is elevated in 30-40% of all hypertensive patients; specifically, the low-renin subset. However, very little is known about the regulation of PHF secretion. Since the classical parathyroid regulator, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), is elevated concurrent with or preceding the development of low-renin hypertension and elevated plasma PHF, we hypothesized that 1,25-(OH)2D3 would stimulate PHF release. PTG organ and cell cultures derived from spontaneously hypertensive rats (SHR) and the normotensive genetic control Wistar Kyoto (WKY) rats, were exposed to various vitamin D3 metabolites and PHF release measured by ELISA. Expression of the classical nuclear vitamin D receptor (1,25VDRnuc), and the putative membrane bound vitamin D receptor (1,25VDRmem) were assessed by western blot using selective antibodies. 1,25-(OH)2D3 rapidly stimulated PHF release from PTG cell cultures with enhanced sensitivity in SHR compared to WKY cultures indicated © 2003 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.

POSTERS: Vasoactive Hormones/Autacoids

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by a leftward shift in the dose-response curve (EC50: 1.52 x 10-8 M for SHR versus 9.64 x 10-8 M for WKY after 30 minutes). PTG organ cultures exhibited a similar sensitivity. The vitamin D3 metabolite 24,25(OH)2D3 had the converse effect, it suppressed basal PHF release. The vitamin D3 analog “BT”, an agonist for 1,25VDRnuc, was without effect suggesting a 1,25VDRnuc-independent mechanism and potential involvement of the 1,25VDRmem. Interestingly, protein expression of the 1,25VDRmem was increased in SHR vs. WKY parathyroid cells. 1,25-(OH)2D3 stimulated release of PHF from parathyroid glands at near physiological concentrations. Stimulation of release was detected after short exposure to 1,25-(OH)2D3. SHR parathyroid glands exhibited increased sensitivity to 1,25-(OH)2D3 compared to WKY parathyroid glands, and had increased expression of 1,25VDRmem. The stimulation of release may occur through actions on the putative 1,25VDRmem . These results support the idea that 1,25-(OH)2D3 may contribute to elevated plasma PHF in the SHR. Key Words: parathyroid hypertensive factor, vitamin D3, parathyroid gland

P-364 NEUROHORMONAL FACTORS IN NORMOTENSIVE SUBJECTS. COMPARASION BETWEEN SUPINE, ACUTE AND PROLONGED ORTHOSTATIC STIMULUS Joao P Freitas, Rosa M Santos, Frans Boomsma, Anton H Meiracker, Emilia Teixeira, Mario J Carvalho, Cassiano A Lima. Centro de Estudos da Funcao Autonomica, Hospital de sao Joao, Porto, Portugal; Department of Internal Medicine, Erasmus University Rotterdam, Rotterdam, Netherlands; Faculdade de Medicina, Universidade do Porto, Porto, Portugal. Prolonged orthostatic stimulus induce a huge change on hemodynamic and autonomic nervous system (ANS) function. Neurohormonal activation difference between acute orthostatic stimulus and prolonged orthostatic stimulus that could trigger the neurocardiogenic response is not clear. The goal of this study was the assessment of neurohormonal behavioural in acute and prolonged orthostatic stimulus. Twelve normotensive subjects (without any medication) underwent were blood sampled at supine rest (S), early passive orthostatic stimulus (first 10 min) (T1) and prolonged (⬎ 40 min) orthostatic stimulus (T2). We measured atrial and brain neuropeptides (ANP and BNP) and cathecolamins (Noradrenaline (NOR), Adrenaline (ADR) and Dopamine (DOP)). ANP was 7.0⫾4.3 pmol/l in S, 7.3⫾5.1 in T1 and 4.6⫾2.8 in T2 (ns and p⬍0.05). BNP was 1.9⫾1.6 pmol/l in S, 1.7⫾1.5 in T1 and 1.4⫾1.3 in T2 (p⬍0.05 and p⬍0.05). NOR was 172⫾92 pg/ml in S, 378⫾216 in T1 and 402⫾183 in T2 (p⬍0.01 and ns). ADR was 10.4⫾3.8 pg/ml in S, 22.2⫾9.3 in T1 and 44.4⫾26.0 at T2 (p⬍0.01 and p⬍0.01). DOP was 7.8⫾4.8 pg/mol, 7.6⫾2.1 at T1 and 7.0⫾2.3 in T2 (ns and ns). Neurohormonal behaviour had mixed responses with orthostatic stimulus. Natriuretic neuropeptides decreased with prolonged orthostatic stimulus probably by the progressive hypovolemia provoked by prolonged orthostatic stimulus. Dopamine levels did not change with any orthostatic stimulus. Noradrenaline and adrenaline had a huge rise with acute orthostatic stimulus although, only adrenaline had a further rise with prolonged orthostatism. Neurohormon behaviour with prolonged orthostatic stimulus could help to clarify the pathophysiology of neurocardiogenic events. Key Words: tilt, natriuretc factors, cathecolamine 0895-7061/03/$30.00