Effect Of Bovine Parathyroid Hormone Research Articles

Bovine parathyroid hormone enhances osteoclast bone resorption by modulating V-ATPase through PTH1R.

The vacuolar-type H+ adenosine triphosphatase (V-ATPase) plays an important role in cellular acidification and bone resorption by osteoclasts. However, the direct effect of bovine parathyroid hormone (bPTH) on V-ATPase has not yet been elucidated. The aim of the present study was to assess the effects of bPTH on V-ATPase and osteoclasts. Osteoclasts from bone marrow (BM)-derived monocytes of C57BL/6 mice were cultured with or without bPTH. The mRNA and protein expression levels of the V-ATPase a3-subunit and d2-subunit (by RT-qPCR and western blot analysis), V-ATPase activity (using the V type ATPase Activity Assay kit) and the bone resorption function of osteoclasts (by bone resorption assay) were examined following treatment with various concentrations of bPTH (0.1, 1.0, 10 and 100 ng/ml) alone or with bPTH and its inhibitor, bafilomycin A1. Furthermore, the expression of parathyroid hormone (PTH) receptors in osteoclasts was also detected. The results revealed that the mRNA and protein expression levels of V-ATPase a3-subunit and d2-subunit increased in a dose-dependent manner, paralleling the level of bPTH present. In addition, an increase in the concentration of bPTH was accompanied by the increased resorption capability of osteoclasts, whereas bone resorption was inhibited in the presence of bafilomycin A1. In addition, we confirmed the existence of parathyroid hormone 1 receptor (PTH1R) in osteoclasts using three different methods (RT-qPCR, western blot analysis and immunofluorescence staining). We found that bPTH enhanced the bone resorption capability of osteoclasts by modulating the expression of V-ATPase subunits, intracellular acidification and V-ATPase activity. Thus, we propose that PTH has a direct effect on osteoblasts and osteoclasts, and that this effect is mediated through PTH1R, thus contributing to bone remodeling.

Resistance to parathyroid hormone-induced inhibition of inorganic phosphate transport in opossum kidney cells cultured in low inorganic phosphate medium.

Renal resistance to the phosphaturic action of parathyroid hormone (PTH) is observed during dietary deprivation of inorganic phosphate (Pi) in vivo. In the present work, the influence of short (3 h)- or long (72 h)-term deprivation of Pi on the effect of bovine PTH (bPTH(1-34)) on both Na-dependent Pi transport and cyclic AMP(cAMP) production was examined in cultured opossum kidney epithelium. Na-dependent Pi transport increased by 100% in cells exposed to low Pi medium containing no Pi (LPM) for 3 h, as compared with transport in high Pi medium containing 2 mmolPi/l (HPM). In response to a submaximal dose (1 nmol/l) of bPTH(1-34), Na-dependent Pi transport was similarly inhibited by about 40% in LPM and HPM. This inhibition was preceded by increased cAMP production which was identical in LPM and HPM. In opossum kidney cells exposed for 72 h to LPM, Na-dependent Pi transport was also increased by 100% compared with that in HPM. However, bPTH(1-34) added at 1 nmol/l did not induce any significant change in Na-dependent Pi transport or cAMP. Stimulation of cAMP could only be elicited at bPTH(1-34) concentrations higher than 1 nmol/l. Such a reduced cAMP response was also observed with forskolin in cells incubated for 72 h in LPM. The cellular resistance to the generation of cAMP was associated with a significantly lower level of ATP in cells cultured for 72 h in LPM compared with ATP levels in HPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum

The effects of bovine parathyroid hormone (bPTH 1–84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 n M bPTH 1–84, there was an ∼2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 m M isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 n M bPTH 1–84 to tissues pretreated with IBMX resulted in an ∼3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 n M bPTH 1–84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH 1–84 but no change in the medium [cAMP]. In tissues pretreated with 1 m M IBMX the response to 10 n M bPTH 1–84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 n M bPTH 1–84. These results suggest the presence ofan adenylate cyclase that can be activated by a mammalian bPTH 1–84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.

Changes in renal function of the chicken associated with calcitonin and parathyroid hormone

We have studied the effects of bovine parathyroid hormone (PTH) and salmon calcitonin (CT) on plasma calcium (Ca) and phosphate levels and on renal function in intact and parathyroidectomized (PTX) chickens, using a simple technique that separates urine from feces. In immature males and egg-laying females, the intravenous injection of PTH (10–20 IU/kg body wt) caused a transient fall in plasma Ca and phosphate levels, which was followed by an increase to a maximal level 20–30 min after the injection. By contrast, adult cockerels seldom showed any variation in plasma Ca or phosphate levels. In all birds, treatment with PTH increased glomerular filtration rate, urine flow rate, phosphate clearance, and Ca clearance, although this peaked later and remained elevated longer than did the phosphate clearance. The infusion of CT (1–20 U/kg over 30 min) caused a significant decrease in plasma Ca only in PTX or partially PTX chickens. All birds showed an increased GFR, urine volume, and urinary Ca excretion. In parathyroid-intact birds, the renal clearance of phosphate also increased, but peaked later and remained high longer than did Ca clearance, suggesting an increased endogenous PTH secretion. We conclude that plasma Ca and phosphate homeostasis in the chicken, as in the mammal, depends on a balanced release of PTH and CT. However, in the chicken, the response time is faster, thus minimizing the fluctuations in plasma Ca and phosphate levels resulting from exogenous hormone administration. This rapid homeostatic response is less effective when Ca demands by the body are high as in the growing or laying bird.

Hypocalcemic effects of bovine parathyroid hormone (1-34) and Stannius corpuscle homogenates in teleost fish adapted to low-calcium water.

Injections of bovine parathyroid hormone (PTH 1-34) and homogenates of corpuscles of Stannius produce hypocalcemia in male killifish and tilapia adapted to calcium-deficient seawater or fresh water, respectively. In fish from water with normal calcium concentrations no effects are noticeable. These results suggest similarity in bioactivity between PTH, the hypercalcemic hormone of terrestrial vertebrates, and the hypocalcemic factor of the corpuscules of Stannius in teleost fish.

A comparison of the effects of bovine parathyroid hormone 1–34 and methylated prostaglandin E 2 analogues on plasma calcium and inorganic phosphate levels in immature chickens and rats

The relative effects of bovine parathyroid hormone 1–34 (bPTH-(1–34)) and prostaglandin (PG)E 2 analogues, 16,16-dimethyl PGE 2 and 11,16,16-trimethyl PGE 2, on plasma calcium, inorganic phosphate ( P i), and osmotality in 12-day-old chickens were compared, bPTH-(1–34) and 16,16-dimethyl PGE 2 were similarly tested for effects on plasma Ca and P i in immature (3-week-old) rats. PGs administered iv in a saline vehicle (20 μg 16,16-dimethyl PGE 2 or 125 μg 11,16,16-trimethyl PGE 2/100 g body wt, respectively) produced significant hypercalcaemic effects in chicks compared with vehicle-injected controls at times ranging from 15 to 19 min, similar to those obtained with bPTH-(1–34) (3.3 μg/100 g body wt). The PGE 2 analogues injected in chicks also produced significant increases in plasma P i compared with controls at 15 and 45 min, while those related with PTH showed smaller, nonsignificant plasma P i responses. Little change in plasma osmolality was seen in chicks following injection with either bPTH-(1–34) or 16,16-dimethyl PGE 2, although a small but significant increase in this parameter was seen in chickes treated with 11,16,16-trimethyl PGE 2. Acute labelling of chicks with 32P incorporated into the 16,16-dimethyl PGE 2 injection vehhicle indicated a decreased exit of P i from the blood as the mechanism underlying the PG-mediated hyperphosphataemic response. This type of response was not seen following mediated hyperphosphataemic response. This type of response was not seen following bPTH-(1–34) injection in chicks. Both 16,16-dimethyl PGE 2 (20 μg/100 g body wt) and bPTH-(1–34) (6.6 μg/100 g body wt) injected iv into rats produced significant hypercalcaemic responses compared with controls at 45 min, although they were small compared to those in chicks. A small but significant hyperphosphataemia was also seen in response to the PG but not to bPTH-(1–34) at 45 min.

Comparative study on the cardiac actions of bovine parathyroid hormone (1–34)

The cardiac stimulatory effects of bovine parathyroid hormone (1–34) [bPTH-(1–34)] were studied on isolated atria and ventricular strips from cobra snakes ( Naja naja), ducklings ( Anas platyrhynchos), pigeons ( Columba livia), Japanese quails ( Coturnix coturnix japonica), bPTH-(1–34) caused positive chronotropism. This suggests that the chronotropic effect of PTH is ubiquitous among the terrestrial vertebrates. Only the atria of cobra snakes and ducklings gave positive inotropic responses to PTH. No ventricular preparations showed significant response to bPTH-(1–34). This suggests the absence of cardiac stimulatory PTH receptors in the ventricles. Dog papillary muscle, however, showed slight but significant responses to bPTH-(1–34). The reason for this discrepancy between the responses of palillary muscles and the ventricular strips to PTH is unknown.

Effects of bovine parathyroid hormone and 1,25-dihydroxyvitamin D3 on the production of prostaglandins by cells derived from human bone

Local production of prostaglandins by osteoblasts may be important in controlling the bone resorbing activity of some hormones which have receptors on osteoblasts. We have demonstrated that osteoblast-like cells derived from human bone can incorporate [14C]arachidonic acid into phospholipids and synthesise immunoreactive PGE. Parathyroid hormone increases both the release of incorporated arachidonic acid and the synthesis of PGE. This is the first demonstration of modulation of bone cell prostaglandin synthesis by a bone resorbing hormone.

Relaxation of bovine, porcine and human brain arteries by parathyroid hormone

We have studied the relaxant effect of bovine parathyroid hormone (bPTH) on helical strips of branches of bovine and human middle cerebral arteries and bovine and porcine basilar arteries. All arteries were studied after contraction with prostaglandin (PG) F 2α or KCl. In the case of all arteries contracted with PGF 2α, the ED 50 of PTH vasorelaxation related to maximal vasorelaxation induced by papaverine ranged from 9 to 14 nM for BPTH- (1–34) and 100 to 220 ng/ml for native bPTH-(1–84). The PTH inhibitor, [Nle 8, Nle 18, Tyr 34] bPTH-(3–34) amide, attenuated the vasorelaxant effect of both bPTH-(1–34) and bPTH-(1–84). The vasorelaxant effects of PTH which we have observed in this study are consistent with the stimulatory effects of PTH on vascular adenylate cyclase which we had previously reported.

Response of phosphate transport to parathyroid hormone in segments of rabbit nephron.

The effects of bovine parathyroid hormone (PTH) on phosphate transport were examined in proximal convoluted tubules, proximal straight tubules, and cortical collecting ducts isolated from the rabbit kidney. The lumen-to-bath flux of phosphate (JlbPO4) and the bath-to-lumen flux (JblPO4) were measured simultaneously with [33P]phosphate and [32P]phosphate. In the proximal convoluted segments perfused with an ultrafiltrate of normal serum, PTH reduced the fluid absorption rate from 1.21 +/- 0.10 to 0.60 nl/mm-min but did not affect JlbPO4, which averaged 5.45 +/- 0.97 pmol/mm-min, or JblPO4, which was 0.50 +/- 0.08 pmol/mm-min. During perfusion with low bicarbonate-high chloride fluids at pH 7.4, the PTH-induced changes in fluid absorption were eliminated but no change occurred in phosphate transport. On the other hand in proximal straight segments JlbP04 was lower at 2.64 +/- 0.41 pmol/mm-min and was directly inhibited by PTH to 1.90 +/- 0.34 pmol/mm-min (P less than 0.001). Net phosphate transport was not observed in cortical collecting ducts in the presence or absence of PTH. These data suggest that phosphate absorption in the proximal tubule involves more than one transport system, that the effects of PTH on fluid absorption are not interdependent with the effects on phosphate transport, and that the proximal straight tubule appears to be an important site of PTH-sensitive phosphate transport.

Effect of bovine parathyroid hormone 1-34 fragment on renal production and excretion of adenosine 3', 5' monophosphate in man.

Biologically active bovine parathyroid hormone (b PTH) 1-34 fragment infused over one hour in normal subjects produced an immediate and sharp increase in the excreted fractions of filtered bicarbonate, sodium and potassium, followed by the return to pre-infusion levels as soon as the administration of b PTH was stopped. There was a gradual but steady increase in the excreted fraction of filtered phosphate but a decrease in the excreted fraction of filtered calcium and magnesium. The excreted fractions of these ions were still abnormal 150 minutes after the completion of PTH infusion. The urinary excretion of 3'5'-cyclic AMP increased immediately about one hundred-fold but returned rapidly to pre-infusion levels. Urinary clearance of cyclic AMP approximated glomerular filtration rate in control periods and was twenty to thirty times greater during b PTH infusion. In subjects overloaded with bicarbonate, b PTH brought about a decrease in bicarbonate T(m) and the same effects on the urinary excretion of other electrolytes. 3'5'-cyclic AMP excretion was clearly higher in control periods and reached higher levels during b PTH infusion when compared to subjects without an alkaline load. 3'5'-cyclic AMP excretion and fractional clearance were also clearly higher in subjects not given b PTH when control periods were compared to periods with bicarbonate infusion or after acetazolamide administration. During distal blockade obtained by simultaneous administration of chlorothiazide and ethacrynic acid, there was a delay in the rise of 3'5'-cyclic AMP excretion after b PTH administration. It can be concluded from these studies that the pattern of excretion of 3'5'-cyclic AMP is similar to that of bicarbonate, sodium and potassium. The increase of 3'5'-cyclic AMP excretion when urinary pH is above 7 suggests a diffusion trapping mechanism for the secretion into the lumen of this nucleotide. Distal diuretics used in distal blockade did not inhibit 3'5'-cyclic AMP production but delayed its secretion into urine.

Effect of Bovine Parathyroid Hormone 1–34 Fragment on Renal Production and Excretion of Adenosine 3‘, 5’ Monophosphate in Man

Abstract. Biologically active bovine parathyroid hormone (b PTH) 1–34 fragment infused over one hour in normal subjects produced an immediate and sharp increase in the excreted fractions of filtered bicarbonate, sodium and potassium, followed by the return to pre‐infusion levels as soon as the administration of b PTH was stopped. There was a gradual but steady increase in the excreted fraction of filtered phosphate but a decrease in the excreted fraction of filtered calcium and magnesium. The excreted fractions of these ions were still abnormal 150 minutes after the completion of PTH infusion. The urinary excretion of 3'5′‐cyclic AMP increased immediately about one hundred‐fold but returned rapidly to pre‐infusion levels. Urinary clearance of cyclic AMP approximated glomerular filtration rate in control periods and was twenty to thirty times greater during b PTH infusion. In subjects overloaded with bicarbonate, b PTH brought about a decrease in bicarbonate Tm and the same effects on the urinary excretion of other electrolytes. 3'5′‐cyclic AMP excretion was clearly higher in control periods and reached higher levels during b PTH infusion When compared to subjects without an alkaline load. 3'5′‐cyclic AMP excretion and fractional clearance were also clearly higher in subjects not given b PTH when control periods were compared to periods with bicarbonate infusion or after acetazolamide administration. During distal blockade obtained by simultaneous administration of chlorothiazide and ethacrynic acid, there was a delay in the rise of 3'5′‐cyclio AMP excretion after b PTH administration. It can be concluded from these studies that the pattern of excretion of 3'5′‐cyclic AMP is similar to that of bicarbonate, sodium and potassium. The increase of 3'5′‐cyclic AMP excretion when urinary pH is above 7 suggests a diffusion trapping mechanism for the secretion into the lumen of this nucleotide. Distal diuretics used in distal blockade did not inhibit 3'5′‐cyclic AMP production but delayed its secretion into urine.