Parathyroid hormone-induced changes in cyclic nucleotide levels during relaxation of the rabbit [correction of rat] aorta

Parathyroid hormone is a potent vasodilator in vivo and relaxes vascular tissue in vitro. Since parathyroid hormone action in kidney and bone is thought to be mediated by stimulation of cellular cyclic AMP production, the present study was designed to monitor changes in cyclic AMP and cyclic GMP in vascular tissue during relaxation by parathyroid hormone. Rabbit aortic strips were quick-frozen at various times after exposure to parathyroid hormone and the percent relaxation and cyclic nucleotide levels were determined. Cyclic AMP concentrations were elevated about 3-fold within 30 seconds after treatment with hormone. This corresponded to a 10% relaxation of the norepinephrine-contracted tissue. After five minutes, cyclic AMP was still elevated 2-fold above basal and the relaxation response was maximal (36%). The cyclic AMP and relaxation responses to parathyroid hormone were markedly potentiated by forskolin or methylisobutylxanthine. Parathyroid hormone produced a small but significant increase in cyclic GMP concentrations only at early time points whereas sodium nitroprusside substantially increased cyclic GMP and relaxed the strips at all times studied. The increase in cyclic AMP levels after exposure to parathyroid hormone occurred prior to or coincident with the onset of relaxation of the aortic strips. These findings are supportive of the hypothesis that the vascular actions of parathyroid hormone involve cyclic AMP.     

Correlation of structural changes in parathyroid hormone with its vascular action

The analysis of the spectrum of circular dichroism (CD) of methionine-oxidized bovine parathyroid hormone peptide, bPTH(1–34) revealed that approximately 43% of the orderly conformation (-helix and β-sheet) was converted into random coil structure. This peptide failed to elicit any hypotensive response in rats at any of the tested doses from 0.01 to 0.05 mg/ml. The blue shift of tryptophan fluorescence and the increase in the fluorescence intensity of the fluorescence probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) bound to the oxidized peptide indicated that the more hydrophobic environment was generated in the tryptophan domain as well as the molecule as a whole when the methionines in the peptide were oxidized. Modification of arginine with 1,2-cyclohexanedione (CHD) reduced 30% to 50% of the hypotensive action of the peptide hormone. Similar results in the increase of hydrophobicity of the arginine-modified peptide were also observed. These studies suggest that the conformational changes due to the methionine oxidation or arginine modification may be related to the inactivation of the vascular activity of bPTH(1–34).     

Functional parathyroid hormone receptors are present in an umbilical vein endothelial cell line

Acute parathyroid hormone exposure induces vascular smooth muscle relaxation. In contrast, continuous infusion of parathyroid hormone leads to vasoconstriction and an elevation of blood pressure. Despite the known effects of parathyroid hormone on vascular smooth muscle, possible direct effects on the vascular endothelium have not previously been investigated. Using a human umbilical vein endothelial cell line, we found that parathyroid hormone increased both intracellular calcium and cellular cAMP content in these endothelial cells. Furthermore, exposure of these cells to increasing concentrations of parathyroid hormone stimulated both [(3)H]thymidine incorporation and endothelin-1 secretion. Parathyroid hormone/parathyroid hormone-related peptide receptor mRNA could be detected at low levels in these cells. In summary, these data demonstrate that endothelium-derived cells contain functional parathyroid hormone receptors. The potential physiological role of these receptors remains to be determined.     

Demonstration that cyclic adenosine 3',5'-monophosphate mediates the lipolytic action of parathyroid hormone.

Studies were carried out with rat epididymal fat pads first to compare the effects of the synthetic N-terminal 1-34 peptide of bovine parathyroid hormone and of the native hormone to determine whether this portion of the molecule is responsible for the lipolytic action of the hormone and second to determine whether this biologic action of parathyroid hormone is mediated by cyclic adenosine 3',5'-monophosphate. The N-terminal polypeptide was as effective as the native hormone in stimulating lipolysis in the concentration range between 10(-8) M and 10(-6) M. Parathyroid hormone stimulated lipolysis by isolated fat cells. The concentration of cyclic adenosine 3',5'-monophosphate in the fat pads was significantly increased by the hormone (10(-6)M). Lipolytic stimulation by parathyroid hormone (10(-6)M) was diminished by insulin (100 muU/ml) and prostaglandin E1 (1 mug/ml), both of which are known inhibitors of lipolysis. The findings indicate that the amino-terminal 1-34 peptide portion of parathyroid hormone is responsible for the lipolytic action and that this effect is mediated through cyclic adenosine 3',5'-monophosphate.     

Possible role of calcium mediators in parathyroid hormone action on phosphate transport in rabbit renal brush border membrane

The possibility of the involvement of intracellular calcium in the action of parathyroid hormone on phosphate transport in renal brush border membrane was examined. Preincubation of rabbit renal proximal tubules with parathyroid hormone or 8-bromo-cAMP induced a significant inhibition on phosphate uptake by the brush border membrane vesicles isolated therefrom. The addition of intracellular Ca antagonists, trifluoperazine or W-7, to the preincubation medium, alone was without effect on phosphate uptake by the brush border membrane vesicles, but abolished the inhibitory effects of parathyroid hormone and 8-bromo-cAMP.     

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1-34 (hPTH (1-34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.     

Treatment of Paget's Disease of Bone with Mithramycin

We report data from three patients with severe Paget''s disease of bone who were treated with mithramycin.Mithramycin infusion resulted in a fall in plasma calcium, phosphate, alkaline phosphatase, and urinary hydroxyproline excretion. There was an improvement in calcium and phosphorus balance in two of the three subjects studied. A pronounced or complete relief of bone pain occurred in all three.We suggest that mithramycin exerts its beneficial effect in Paget''s disease of bone by stimulating parathyroid hormone release. The parathyroid hormone released has a predominantly anabolic action on bone since its catabolic action is blocked by mithramycin, which inhibits bone resorption.     

Intact and carboxyterminal PTH do not cross the blood-cerebrospinal fluid barrier

In order to examine whether parathyroid hormone (PTH) enters the cerebrospinal fluid (CSF), the blood levels of the hormone were acutely elevated either by infusion of parathyroid extract or by stimulation of the parathyroid glands by hypocalcemia. Despite marked elevations in the blood levels of the hormone, PTH could not be detected in the CSF. The data indicate the intact PTH or its carboxyterminal fragment do not cross the blood-CSF interface of the blood-brain barrier. The results, therefore, suggest that the action of PTH on brain must be mediated by an effect on the blood-brain interface of the blood-brain barrier.     

Dopamine-stimulated parathyroid hormone release in vitro: further evidence for a two-pool model of parathyroid hormone secretion

Bovine parathyroid tissue was placed in an in vitro perifusion system for the study of parathyroid hormone secretion stimulated by low calcium and dopamine. Dopamine caused a transient increase in parathyroid hormone release, while low calcium caused a sustained increase in parathyroid hormone secretion. The dopamine response was similar to that caused by isoproterenol. After parathyroid hormone release had been stimulated by dopamine there was no response to isoproterenol, suggesting they cause the release of the same cellular pool of hormone. Inhibition of protein synthesis with cycloheximide eliminated the response to low calcium, with no effect on dopamine-stimulated parathyroid hormone release. These studies suggest dopamine stimulates the release of a limited quantity storage pool of parathyroid hormone, while low calcium causes a sustained release of hormone by stimulating secretion of newly synthesized hormone. Low calcium has little or no effect on release of the storage granule pool of parathyroid hormone.     

Cyclic AMP and the vascular action of parathyroid hormone

The involvement of tissue cAMP in the vasodilating action of parathyroid hormone (PTH) was investigated. The bovine active fragment bPTH-(1-34) was used in all studies. In anesthetized dogs, theophylline, a phosphodiesterase inhibitor, potentiated the hypotensive action of bPTH-(1-34) at the dose of 1 microgram/kg. The potentiation was related to the dose of theophylline infused. In an in vitro rat tail artery helical strip assay, dibutyryl cAMP produced dose-related relaxation in arginine vasopressin (AVP) constricted blood vessels. bPTH-(1-34) also produced dose-related relaxation in the tail artery constricted by AVP. In the presence of isobutylmethylxanthine, another phosphodiesterase inhibitor, the bPTH-(1-34) dose--response curve was shifted to the left, indicating potentiation. Imidazole, which has phosphodiesterase stimulating activity, significantly decreased the in vitro vasorelaxing effect of bPTH-(1-34). In addition, bPTH-(1-34) increased significantly the rat tail artery cAMP content. b-PTH-(1-34) oxidized with hydrogen peroxide lost its vasorelaxing activity and was also ineffective in increasing the tail artery cAMP content. All these data strongly suggest that cAMP may be involved in eliciting the vasorelaxing action of bPTH-(1-34).     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

Influence of parathyroid hormone and heparin on tooth development in mice homozygous for the microphthalmia mutation]

In mice, homozygous by microphthalmia (mi/mi) gene the disturbance of dental development was due to defects in dental pulp and to the absence of bone resorpion. Administration of heparin or parathyroid hormone stimulated dental eruption in mi/mi mice. Following combined action of heparin and parathyroid hormone the number of erupted teeth was practically the same as after the action of heparin alone. Possibly the level of heparin is insufficient, but secretion of parathyroid hormone is unimpaired in mi/mi mice.     

Responsiveness of mouse calvaria to parathyroid hormone after explant cryopreservation: 45Ca release in vitro

Newborn mouse calvaria prelabeled with 45Ca and cryopreserved at -196 degrees C in serum-free medium containing dimethylsulfoxide were compared to unpreserved explants for response to parathyroid hormone during subsequent culture. After short-term cryopreservation followed by rapid thawing, the viable explants continued to release 45Ca to the culture medium but additions of parathyroid hormone to the medium did not cause increased bone resorption. The data suggest that cryopreservation and thawing impairs mechanisms responsible for parathyroid hormone action on bone cells.     

The calcium-mediated promotion of mitotic activity in rat thymocyte populations by growth hormone, neurohormones, parathyroid hormone and prolactin

Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone. A possible common mechanism of action of these hormones on mitotically competent cells is discussed.     

Parathyroid hormone inhibition of phosphate transport in renal brush border vesicles from phosphate-depleted dogs

Dietary phosphate (P_i) restriction increases renal P_i reabsorption and induces resistance to the phosphaturic action of parathyroid hormone. Na⁺-gradient-stimulated P_i transport in membrane vesicles isolated from the renal brush border of experimental animals has been shown to parallel changes in renal P_i reabsorption induced by dietary P_i restriction and in vivo administration of parathyroid hormone. Dietary P_i restriction has been shown to markedly inhibit the phosphaturic response to parathyroid hormone in rats and dogs. Parathyroid hormone has been reported not to decrease the Na⁺-gradient-stimulated transport of P_i in brush border membrane vesicles isolated from dietary P_i restricted rats unless the rats were administered an acute P_i load prior to killing, however, thyroparathyroidectomy of rats fed a low P_i diet has been reported to increase Na⁺-gradient-stimulated P_i transport. Using the dietary P_i restricted dog, we demonstrated no significant decrease in renal reabsorption of P_i in response to parathyroid hormone administration. However, significant decreases in P_i transport in brush border membrane vesicles isolated from the kidneys of dietary P_i restricted dogs were observed in response to in vivo parathyroid hormone administration. These data demonstrate that the resistance to the phosphaturic action of parathyroid hormone observed in vivo does not include resistance to the inhibitory effect of parathyroid hormone on P_i transport in brush border membrane vesicles. Thus, the data suggest that parathyroid hormone continues to alter P_i transport characteristics of the brush border membrane in states of P_i depletion despite the resistance to parathyroid hormone seen in vivo.     

The passive role of calcium in hypertension: a position statement as of August 20, 1985

Epidemiologic data suggest that calcium intake is lower in hypertensive individuals than in normotensive individuals. Moreover, hypertensive patients and rats excrete more calcium in their urine than normotensives, and laboratory studies (low ionized calcium, high parathyroid hormone (PTH)) suggest that the hypertensive is somewhat calcium depleted. However, both the low ionized calcium and elevated PTH should tend to lower pressure rather than raise it, leaving only the presumed and poorly documented natriuretic effect of calcium as a hypotensive mechanism. The evidence is better for a high sodium intake producing a conditioned deficiency of calcium by increasing calcium excretion than for a low intake of calcium raising blood pressure.     

Effects of calcium and parathyroid hormone on prostacyclin synthesis by vascular tissue

Prostacyclin generation by rat aortic rings was studied at different calcium concentrations. Extracellular calcium influenced prostacyclin synthesis, as reflected by the release of 6-keto-PGF1 alpha into the medium, in a concentration-dependent fashion. Calcium levels beyond the physiological range (1.12-1.25 mM unbound calcium) markedly stimulated 6-keto-PGF1 alpha production when compared with calcium-free solutions. On the other hand, addition of purified parathyroid hormone did not change 6-keto-PGF1 alpha production at any calcium concentration tested. These data suggest that parathyroid hormone has no direct effect on prostacyclin synthesis by vascular tissue, although it might influence prostacyclin generation through changes in extracellular calcium levels.     

Endocytosis and phosphate transport in OK epithelial cells

Endocytosis was studied in OK epithelial cells, an established cell line from opossum kidney. The presence of fluid-phase endocytosis in these cells was demonstrated by measuring cell uptake of lucifer yellow and horseradish peroxidase. The intracellular distribution of lucifer yellow fluorescence was consistent with uptake by endocytosis. Endocytosis was inhibited in medium made hyperosmolar by addition of sucrose. In hyperosmolar medium the action of parathyroid hormone on Na+/phosphate cotransport was significantly diminished. We suggest that an intact endocytic mechanism is required for the full inhibitory effect of parathyroid hormone on Na+/phosphate cotransport.     

Parathyroid hormone and calcium entry blockade in a vascular tissue

The mechanism of the vasorelaxing action of a synthetic parathyroid hormone fragment, bPTH-(1-34), was studied. Rat tail artery helical strips were constricted in vitro with potassium chloride (6 x 10(-2) M), arginine vasopressin (2 mU/ml) or BAY-K-8644 (3 x 10(-7) M in the presence of 1.5 x 10(-2) M KCl). bPTH-(1-34) was able to relax the constricted tissue or to inhibit the constriction. All three constricting agents increased calcium uptake by the vascular tissue as determined by the measurement of the low-affinity lanthanum-resistant pool of calcium. Such increases in calcium uptake were significantly reduced by bPTH-(1-34). These data suggest that PTH may be a natural circulating hormone or chemical capable of inhibiting calcium entry in a vascular tissue.     

Increased calcium intake reduces plasma cholesterol and improves vasorelaxation in experimental renal failure

Chronic renal failure (CRF) is associated with abnormal lipid metabolism and high prevalence of vascular complications. Calcium salts are commonly used in CRF as phosphate binders. Increased calcium intake may also lower plasma cholesterol and beneficially influence vascular tone. Therefore, we investigated the influence of increasing dietary calcium from 0.3% to 3.0% for 8 wk after 5/6 nephrectomy (NTX) on plasma cholesterol and mesenteric resistance vessel tone in male Sprague-Dawley rats. The groups were Sham, Sham-Calcium, NTX, and NTX-Calcium (n = 10-11). Blood pressure was modestly elevated after NTX, whereas the plasma creatinine, urea nitrogen, phosphate, and parathyroid hormone levels were clearly increased. The high-calcium diet suppressed plasma phosphate and parathyroid hormone but was without effect on blood pressure. The NTX resulted in 1.6-fold elevation in plasma total cholesterol and 40% reduction in high density-to-low density lipoprotein ratio (HDL/LDL). However, the lipid profile in NTX rats on the high-calcium diet did not differ from sham-operated controls. The endothelium-mediated relaxations induced by acetylcholine were impaired in NTX rats, whereas the response was normalized by a high-calcium diet. No differences in vasorelaxations by the endothelium-independent vasodilator nitroprusside were detected. In conclusion, improved vasorelaxation after a high-calcium diet could be due to reduced plasma total cholesterol and ameliorated HDL/LDL ratio, although decreased plasma phosphate and parathyroid hormone may also play a significant role in the vascular effects of increased calcium intake.