Production of prostaglandins by dispersed cells and fragments from bovine parathyroid glands

We studied the production of prostaglandins by fragments and dispersed cells from bovine parathyroid glands. Fragments released 138 +/- 19 (SE), 132 +/- 21, 4.3 +/- 0.5, and 13 +/- 6.6 pg/mg/h of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and thromboxane B2, respectively (n = 7 - 26), while dispersed cells released 414 +/- 110, 22 +/- 7.3, 27 +/- 3.8, and 29 +/- 11 pg/10(6) cells/h, respectively, of the same compounds (n = 6 - 25). Indomethacin (1 microgram/ml) inhibited the release of 6-keto-PGF1 alpha by 80-90% in fragments and cells, while mellitin stimulated release of this prostaglandin, suggesting de novo synthesis of prostaglandins in these preparations. Calcium stimulated production of 6-keto-PGF1 alpha by 1.3-fold in cells and 2.6-fold in fragments and also enhanced production of PGF2 alpha by 1.9-fold in fragments. Isoproterenol, on the other hand, had no effect on production of 6-keto-PGF1 alpha in either preparation. These results demonstrate that parathyroid tissue as well as parathyroid cells per se produce a variety of prostaglandins. We have previously shown that PGE2 and PGF2 alpha modulate cAMP accumulation and PTH release in dispersed bovine parathyroid cells. The role of the endogenous production of prostaglandins by the parathyroid gland in the acute or chronic regulation of parathyroid function, however, remains to be determined.     

Paradoxical effects of K+ and D-600 on parathyroid hormone secretion and cytoplasmic Ca2+ in normal bovine and pathological human parathyroid cells

The effects of K+ and the Ca2+ channel blocker D-600 on parathyroid hormone (PTH) release and cytoplasmic Ca2+ activity (Ca2+i) were measured at different Ca2+ concentrations in dispersed parathyroid cells from normal cattle and from patients with hyperparathyroidism. When the extracellular Ca2+ concentration was raised within the 0.5-3.0 mM range Ca2+i increased and PTH secretion was inhibited. There was also a stimulatory effect of Ca2+ on secretion as indicated by a parallel decrease of Ca2+i and PTH release when extracellular Ca2+ was reduced to less than 25 nM. Addition of 30-50 mM K+ stimulated PTH release and lowered Ca2+i. The effect of K+ was less pronounced in the human cells with a decreased suppressability of PTH release. The Ca2+ channel blocker D-600 had no effect on Ca2+i and PTH release in the absence of extracellular Ca2+. However, at 0.5-1.0 mM Ca2+, D-600 increased Ca2+i and inhibited PTH release, whereas the opposite effects were obtained at 3.0 mM Ca2+. The transition from inhibition to stimulation occurred at a higher Ca2+ concentration in the human cells and the right-shift in the dose-effect relationship for Ca2+-inhibited PTH release tended to be normalized by D-600. It is suggested that K+ stimulates PTH release by increasing the intracellular sequestration of Ca2+ and that the reduced response in the parathyroid human cells is due to the fact that Ca2+i already is lowered. D-600 appears to have both Ca2+ agonistic and antagonistic actions in facilitating and inhibiting Ca2+ influx into the parathyroid cells at low and high concentrations of extracellular Ca2+, respectively. D-600 and related drugs are considered potentially important for the treatment of hyperparathyroidism.     

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.     

Effect of parathyroid hormone on renin secretion

The ability of parathyroid hormone (PTH) to increase renin secretion was investigated in pentobarbital-anesthetized dogs. An intravenous infusion of bovine PTH 1-34, at the dose of 0.028 microgram/kg-1 min-1 increased renin secretion by 149% (501 +/- 105 to 1249 +/- 309 ng hr-1 min-1); renin secretion returned to control values during the recovery period. In order to determine whether PTH acted directly on the kidney to increase renin secretion, PTH was infused into the right renal artery at doses of 0.0014 to 0.0028 microgram/kg-1 min-1 and renin secretion from the right kidney was compared to that from the left (control) kidney. Renin secretion from the right (PTH-infused) kidney was not greater than control values for that kidney or different from the renin secretory rate of the left (control) kidney. In contrast, the excretion rates of both phosphate and sodium from the right kidney were greater than control values and from the excretion rates of the left kidney. These data suggest that PTH, while acting directly on the kidney to increase phosphate and sodium excretion, does not elevate renin secretion by a direct renal action.     

Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.     

Direct inhibitory effect of amino-terminal parathyroid hormone fragment [PTH(1-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro

We have examined the possibility of direct inhibitory effect of PTH(1-34) on PTH secretion in bovine parathyroid cells. As low as 10(-12) M PTH(1-34) completely inhibited low calcium (0.5 mM Ca2+)-stimulated PTH secretion by these cells. In the presence of 1.25 mM Ca2+, 10(-12) M PTH(1-34) inhibited PTH secretion by about 14.3% of the basal value, while 10(-11) M or higher concentration of PTH(1-34) showed potent inhibitory effects equivalent to the inhibitory action of high calcium concentration (2.5 mM Ca2+) on PTH secretion. At 2.5 mM Ca2+, as much as 10(-9) M PTH(1-34) failed to inhibit PTH secretion further. These results suggest that PTH(1-34) might directly, not via calcium concentration, inhibit PTH secretion by parathyroid cells and that a cooperative mechanism could exist between calcium and PTH(1-34) to inhibit PTH secretion.     

Suppression of BRCA1 sensitizes cells to proteasome inhibitors

BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity.BRCA1 is an important tumor suppressor gene whose germ-line or somatic inactivation is implicated in a significant number of breast and ovarian cancers.¹ Human BRCA1 encodes an 1863 amino-acid-long protein with a RING-finger domain at the N terminus and two BRCT domains located at the C terminus.^{2, 3} BRCT domains mediate interaction with phosphorylated proteins such as Abraxas, BACH1, CtIP and others involved in sensing DNA damage and assembly of the BRCA1-associated genome surveillance complex at sites of DNA breaks.⁴ The RING domain constitutively interacts with the BRCA1-associated RING domain protein (BARD1), forming a heterodimer having an E3 ubiquitin ligase activity.⁵ Ubiquitination of target proteins, including cell cycle or DNA repair-regulating proteins (e.g. CtIP (RBBP8), nucleophosmin (NPM1, B23), claspin (CLSPN) and others), occurs either at Lys48 residue of the ubiquitin leading to the 26S proteasome-mediated degradation of target proteins or at Lys6 or Lys63 having a trafficking and signaling role.⁶ A serine cluster coiled-coil domain spanning amino acids 1280–1524 contains multiple phosphorylation sites for ATM and ATR kinases activated by DNA damage.⁷ The same region also binds PALB2 protein linking BRCA1 to another major breast cancer predisposition gene BRCA2.⁸The most prominent function of BRCA1 is associated with its role in repair of DNA damage, particularly of double-stranded DNA breaks (DSBs), one of the most severe types of DNA lesions.⁹ BRCA1 is recruited to sites of DNA damage via a series of phosphorylation and ubiquitination events, where it serves as a binding scaffold for other DNA repair proteins,^{10, 11} ubiquitinates claspin, cyclin B and CDC25C, triggering cell cycle arrest to allow time for repair,¹² and facilitates BRCA2-mediated loading of RAD51 recombinase to enable the homologous recombination (HR) mechanism of DNA repair.⁹ In addition, BRCA1 may contribute to maintaining genome integrity by stabilizing the heterochromatin structure via ubiquitination of histone H2A.¹³ BRCA1 is also required for centrosome-dependent and -independent mitotic spindle formation, providing another route, by which loss of BRCA1 could promote chromosome instability and tumor formation.^{14, 15}Such a critical role of BRCA1 in DNA repair is exploited therapeutically. DNA-damaging agents, particularly DNA-crosslinking agents such as platinum-containing drugs, or ionizing radiation lead to the accumulation of DNA breaks requiring HR for repair and, therefore, are particularly toxic to BRCA1-deficient tumor cells.¹⁶ Pharmacological inhibitors of poly-(ADP-ribose) polymerases (PARPs) selectively kill BRCA1-deficient cells owing to defective HR, functioning as a back-up repair mechanism in the absence of the PARP-mediated repair of single-stranded DNA breaks.¹⁷ However, multiple mechanisms allow BRCA1-deficient cells to develop resistance to these drugs including elevated expression of the efflux transporters pumping the drugs out of the cell, secondary mutations restoring a functional BRCA1 protein and loss of 53BP1 protein, which counteracts BRCA1 and HR by blocking resection of DNA ends around the breaks (see Lord and Ashworth¹⁸ for the latest review). Therefore, additional efforts to identify small-molecule agents especially targeting BRCA1 functions unrelated to its DNA repair function are warranted.Here we performed a high-throughput chemical screen of BRCA1-depleted MDA-MB-231 cells using a collection of 198 US Food and Drug Administration (FDA)-approved and experimental drugs. We found that 26S proteasome inhibitors were more toxic to BRCA1 knockdown than control cells. Response of BRCA1-deficient cells to bortezomib involved deregulation of the RB1-mediated cell cycle checkpoint, activation of a noncanonical ERN1-mediated unfolded protein response and 53BP1-related G2/M cell cycle arrest. Our results reveal novel aspects of BRCA1 function unrelated to DNA repair.     

Parathyroid hormone secretion does not respond to changes of free calcium in electropermeabilized bovine parathyroid cells, but is stimulated with phorbol ester and cyclic AMP

The secretion of parathyroid hormone (PTH) is suppressed in bovine parathyroid cells by raised extracellular [Ca2+], and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the release of PTH from cells suppressed by high extracellular [Ca2+]. Extracellular and cytosolic free [Ca2+] are proportionally related in intact cells. To assess the role of cytosolic free [Ca2+] on PTH secretion, bovine parathyroid cells were rendered permeable by brief exposure to an intense electric field. PTH secretion was comparable at 40 nM, 500 nM, 5 microM, 28 microM, 0.5 mM and 2 mM [Ca2+] (release of total cellular PTH 3.7 +/- 0.5%, 3.9 +/- 0.4%, 3.4% +/- 0.3%, 3.9 +/- 0.4%, 3.1 +/- 0.3%, 3.5 +/- 0.7%, respectively), but the secretion was stimulated twofold (P less than 0.05 vs. control) in a dose and ATP dependent manner with TPA (100 nM) and cyclic AMP (1 mM). As a result, free [Ca2+] in the range of those observed in intact cells during regulation of PTH secretion by changes of extracellular [Ca2+] did not affect the release of PTH in permeabilized cells. The [Ca2+] independent stimulation of PTH release by TPA and cyclic AMP indicates that changes of cytosolic free [Ca2+] may represent a secondary event not related to the regulation of PTH secretion.     

Second messenger pathways mediating chicken luteinizing hormone secretion from dispersed pituitary cells.

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Binding of tritiated bovine parathyroid hormone to plasma membranes from bovine kidney cortex.

A membrane fraction enriched in parathyroid hormone (PTH)-sensitive adenylate cyclase and sodium and potassium ion-activated (Na+, K+)-ATPase was prepared from bovine kidney. Tritiated PTH binding to this membrane fraction was dependent on both hormone and membrane protein concentration. Both total and specific binding of the hormone decreased significantly after 5 to 10 min of incubation at 22 degrees. PTH binding was highly specific, being sensitive to inhibition only with active forms of unlabeled hormone (native and 1-34 PTH). Specific binding showed a pH optimum of 7.3 to 7.5. Inhibition of binding of tritiated hormone by unlabeled PTH was also highly effective at pH 6.0, but this apparently specific binding was also inhibited by adrenocorticotropic hormone, insulin, glucagon, and vasopressin. Dissociation of bound hormone was demonstrated, and an apparent dissociation constant of 4.6 X 10(-2) min-1 was obtained. Specific binding was eliminated by pretreatment of the membranes with trypsin. The concentration dependence for inhibition of binding with unlabeled PTH was identical to that for activation of adenylate cyclase in this membrane preparation, and binding was also inhibited by concentrations of calcium in the 0.5 to 2 mM range.     

The degradation of bovine parathyroid hormone by leukocytes

Human peripheral leukocytes catalyse an elastase-like cleavage of bovine parathyroid hormone, with an identical specificity to that previously observed with a neutral proteinase (EC 3.4.21-) isolated from the outer surface of plasma membranes from human leukocytes. Parathyroid hormone is not hydrolysed by human erythrocytes, and polymorphonuclear leukocytes are much more effective in hormone degradation than lymphocytes. Despite the fact that purified human leukocyte granular elastase (EC 3.4.21.37) catalyses an identical cleavage reaction, the hydrolysis observed with intact cells is clearly not due to proteinases secreted by the cells. The reaction is inhibited by human serum and by purified human alpha-1-antitrypsin, but not by antibodies to human leukocyte granular elastase. The possible significance of these phenomena to the in vivo metabolism of parathyroid hormone are discussed.     

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.