Dispersed Bovine Parathyroid Cells Research Articles

Effects of Chai-Qin-Cheng-Qi decoction on regulating protein kinase C pathway in colon smooth muscle cells of rats with acute necrotizing pancreatitis of rats

The sensing of extracellular Ca2+ concentration ([Ca2+]o) and modulation of cellular processes associated with acute or sustained changes in [Ca2+]o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca2+]o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca2+]o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca2+]o required influx of Ca2+through Ni2+-sensitive Ca2+channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca2+]o. Activation of ERK1/2 by high [Ca2+]o was not necessary for the [Ca2+]o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca2+]o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.

Involvement of protein kinase C-alpha and -epsilon in extracellular Ca2+ signalling mediated by the calcium sensing receptor

The sensing of extracellular Ca2+ concentration ([Ca2+]o) and modulation of cellular processes associated with acute or sustained changes in [Ca2+]o are cell-type specific and mediated by the calcium sensing receptor (CaR). [Ca2+]o signalling requires protein kinase C (PKC), but the identity and role of PKC isoforms in CaR-mediated responses remain unclear. Here we show that high [Ca2+]o activated PKC-α and PKC-ε in parathyroid cells and in human embryonic kidney (HEK293) cells overexpressing the CaR (HEK-CaR) and that this response correlated with the CaR-dependent activation of mitogen-activated protein kinases ERK1/2. Activation of ERK1/2 by acute high [Ca2+]o required influx of Ca2+through Ni2+-sensitive Ca2+channels and phosphatidylinositol-dependent phospholipase C-β activity. Inhibition of PKC by co-expression of dominant-negative (DN) mutants of PKC-α or -ε with the CaR attenuated sustained ERK1/2 activation. Overexpression of a PKC phosphorylation site (T888A) mutant CaR in HEK293 cells showed that this site was important for ERK1/2 activation at high [Ca2+]o. Activation of ERK1/2 by high [Ca2+]o was not necessary for the [Ca2+]o-regulated secretion of parathyroid hormone (PTH) in dispersed bovine parathyroid cells. These data suggest that the CaR-mediated [Ca2+]o signal leading to regulated PTH secretion that requires diacylglycerol-responsive PKC isoforms is not mediated via the ERK pathway.

Is the calcium receptor a molecular target for the actions of strontium on bone?

The extracellular calcium-sensing receptor (CaR) plays key roles in maintaining extracellular calcium homeostasis by enabling several of the cells and tissues involved in this process to sense small changes in Ca(2+)(o) and to respond with changes in cellular function that will restore Ca(2+)(o) to its normal level. The chief cells of the parathyroid gland and the thyroidal C-cells, for example, respond to decreases in Ca(2+)(o) with increased secretion of the Ca(2+)(o)-elevating hormone, parathyroid hormone (PTH), and decreased secretion of the Ca(2+)(o)-lowering hormone, calcitonin, respectively. The cells of the renal distal tubule are likewise capable of sensing Ca(2+)(o) and respond to decreases in Ca(2+)(o) with increased tubular reabsorption of Ca(2+) and vice versa, alterations in tubular function that will contribute to normalization of Ca(2+)(o). The skeleton also plays key roles in maintaining Ca(2+)(o) homeostasis and both osteoblasts and osteoclasts can sense Ca(2+)(o), with elevations in Ca(2+)(o) promoting bone formation and inhibiting bone resorption. It has been suggested that Sr(2+) could act on bone via the CaR; however, the molecular mechanisms through which Ca(2+)(o) and Sr(2+)(o) exert these actions on bone cells remain controversial. Therefore, identifying their molecular target(s) would have significant implications for the treatment of bone loss. Ideally, therapies should simultaneously inhibit bone resorption while stimulating bone formation. Administration of strontium produces exactly those effects. Previous studies with dispersed bovine parathyroid cells as well as a preliminary study using CaR-transfected Chinese hamster ovary (CHO) cells indicate that Sr(2+)(o) is an agonist of the CaR, albeit with slightly lower efficacies and potencies than Ca(2+)(o). Given that Sr(2+)(o) is distributed preferentially in bone, therefore, an action of this divalent cation on the CaR in bone cells represents one possible mechanism by which strontium ranelate, a new antiosteoporotic drug, exerts it skeletal actions in vivo.

Biosynthesis and Secretion of Parathyroid Hormone Are Sensitive to Proteasome Inhibitors in Dispersed Bovine Parathyroid Cells

Preproparathyroid hormone (prepro-PTH) is one of the proteins abundantly synthesized by parathyroid chief cells; yet under normal growth conditions, little or no prepro-PTH can be detected in these cells. Although this may be attributed to effective cotranslational translocation and proteolytic processing, proteasome-mediated degradation of PTH precursors may be important in the regulation of the levels of these precursors and hence PTH secretion. The effects of N-acetyl-Leu-Leu-norleucinal, N-acetyl-Leu-Leu-methional, carbobenzoxy-Leu-Leu-leucinal (MG132), benzyloxycarbonyl-Ile-Glu(t-butyl)-Ala-leucinal (proteasome inhibitor I), and lactacystin on the biosynthesis and secretion of PTH were examined in dispersed bovine parathyroid cells. We demonstrate that treatment of these cells with proteasome inhibitors caused the accumulation of prepro-PTH and pro-PTH. Compared with mock-treated cells, the processing of pro-PTH to PTH was delayed, and the secretion of intact PTH decreased in proteasome inhibitor-treated cells. Relieving the inhibition of the proteasome by chasing MG132-treated cells in medium without the inhibitor led to the rapid disappearance of the accumulated prepro-PTH, and the rate of PTH secretion was restored to levels comparable to those in mock-treated cells. Furthermore, overexpression of the Hsp70 family of molecular chaperones was observed in proteasome inhibitor-treated cells, and we show that PTH/PTH precursors interact with these molecular chaperones. These data suggest the involvement of parathyroid cell proteasomes in the quality control of PTH biosynthesis.

G Protein-Coupled Extracellular Ca2+ (Ca2+oRpar;–Sensing Receptor (CaR): Roles in Cell Signaling and Control of Diverse Cellular Functions

Publisher Summary A body of indirect evidence had suggested the presence of a Ca 2+ o -sensing mechanism in parathyroid cells, C-cells, and kidney cells, through which Ca 2+ o could act, in effect, as an extracellular first messenger via its own putative G protein-coupled, cell surface receptor. Such evidence, principally accumulated in studies, involving dispersed bovine parathyroid cells, included some well-defined actions of the elevated Ca 2+ o . The body of evidence, suggesting the existence of a Ca 2+ o -sensing receptor, provides a foundation for the successful use of expression cloning in Xenopm laevis oocytes to isolate a full-length complementary DNA (cDNA), encoding the bovine parathyroid Ca 2+ o -sensing receptor (abbreviated as CaR). Research shows that the CaR provide the molecular basis for a number of the known effects of Ca 2+ o on its target tissues involved in mineral ion homeostasis, especially parathyroid and kidney. Although additional studies are needed to prove its mediatory role, the CaR may likewise participate in the aspects of the control of intestinal function and bone turnover that are relevant to systemic mineral ion metabolism. This receptor also plays key roles in human disorders, such as familial hypocalciuric hypercalcemia (FHH)/neonatal severe hyperparathyroidism (NSHPT), autosomal dominant hypocalcemia, and sporadic hypocalcemia/hypoparathyroidism as well as in the impaired urinary concentrating capacity or, in some cases, frank nephrogenic diabetes insipidus observed in hypercalcemic persons. However, findings of the wide distribution of the CaR in tissues uninvolved in systemic Ca 2+ o homeostasis indicate that the CaR also participates in modulating the neuronal activities in the brain and perhaps intestine as well as a variety of other cellular functions.

Characterization of the phosphatidylinositol-specific phospholipase C isozymes present in the bovine parathyroid and in human kidney HEK293 cells stably transfected with the human parathyroid Ca2+-sensing receptor.

The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.

Inwardly Rectifying K+Channels in Dispersed Bovine Parathyroid Cells

Excitation-secretion coupling in various endocrine cells is dependent on membrane voltage which is controlled by ion channels. In order to characterize and determine the functional significance of voltage-gated ion channels in the parathyroid cell, the patch clamp technique was used in cell-attached and whole cell configurations to study single channel and whole cell currents in dispersed bovine parathyroid cells. Whole cell voltage clamp recordings from dissociated bovine parathyroid cells were obtained in a physiologic solution containing (in mM): 140 NaCl, 5.4 KCl, 2 CaCl2, and 2 MgCl2. The pipette (intracellular) solution contained (in mM) 145 KAsp, 10−5CaCl2, and 2 MgCl2. Currents were recorded in response to 20-mV incremental changes in voltage of 300-ms duration every 3 s from −80 to +40 mV and from −40 to −140 mV. There was a small outward current recorded in response to 300-ms pulses of 20-mV increments from −80 to +40 mV. A large inward current was recorded following hyperpolarization of the parathyroid cell from −40 to −140 mV. The reversal potential for the current was −60 to −65 mV, suggesting that the majority of the current is carried by a channel that is K+selective. Our results suggest that the whole cell currents of dispersed bovine parathyroid cells in physiologic extracellular solution include an in inwardly rectifying K+current which is open at low intracellular calcium concentration. This inwardly rectifying K+channel is likely to play a major role in maintaining negative membrane potential by opposing calcium-induced depolarization of the parathyroid cell and, as a result, may have an important role in regulation of PTH secretion.

A direct effect in vitro of phosphate on PTH release from bovine parathyroid tissue slices but not from dispersed parathyroid cells

Phosphate retention has long been considered to be of importance for the pathogenesis of secondary hyperparathyroidism in chronic renal failure. Hyperphosphatemia in vivo is associated with alterations of calcium and vitamin D levels, both of which are known to alter the parathyroid hormone (PTH) release independently. We have investigated the direct effect of phosphate on PTH release in vitro using two different preparations of bovine parathyroid tissue: Acutely dispersed bovine parathyroid cells and tissue slices of 0.5 x 0.5 mm were prepared from bovine parathyroid glands. Parathyroid dispersed cells and tissue slices were incubated for 4 h in media containing normal phosphate (1.0 mM) or high phosphate (3.5 mM). High phosphate induced a significant (P < 0.01) increase in PTH release in the preparation of tissue slices, but not in preparations of dispersed cells. The 4 h incubation in high phosphate medium did not change the set-point for calcium. Bovine parathyroid tissue slices incubated in increasing phosphate concentrations from 1.0 to 3.5 mM and with a fixed calcium concentration of either 0.8, 1.2 or 1.8 mM responded with a dose dependent stimulation of PTH release. The degree of stimulation of release by high phosphate (3.5 mM), was significantly (P < 0.05) higher at low calcium levels (0.8 mM), 172% above baseline value (1.0 mM phosphate) as compared to high calcium levels (1.8 mM), 139% above baseline values. This study shows that phosphate directly stimulates the PTH release in bovine parathyroid glands, and that this effect is only seen in preparations of parathyroid tissue slices and not in preparations of dispersed cells. This indicated that maintenance of near normal architecture of the parathyroid glands is essential in order to elicit the effect of high phosphate on the PTH release.

Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells

Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells

Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells.

To separate the role of changes in parathyroid diacylglycerol (DG) from other effects of extracellular calcium, we studied the effect of inhibition of DG metabolism on PTH secretion and cellular DG content in acutely dispersed bovine parathyroid cells. R 59 022, an inhibitor of DG kinase, increased cellular DG, but significantly decreased PTH secretion. Particulate protein kinase C (PKC) activity decreased in bovine parathyroid cells incubated at high extracellular calcium or in the presence of R 59 022, which is the opposite of what was observed in the presence of the phorbol ester, phorbol myristate acetate. Sphinganine, a normal cellular product that is a known inhibitor of PKC, significantly inhibited PTH secretion at low extracellular calcium, but had no significant effect at normal or high extracellular calcium. We then measured sphingosine in bovine parathyroid cells incubated with high extracellular calcium or R 59 022. Both conditions were associated with significant elevations of cellular sphingosine. These studies suggest that inhibition of PTH secretion and PKC activity by enhanced cellular DG may result from the activation of an inhibitory second messenger pathway involving the sphingoid lipids.

The reduced responsiveness of cultured bovine parathyroid cells to extracellular Ca2+ is associated with marked reduction in the expression of extracellular Ca(2+)-sensing receptor messenger ribonucleic acid and protein.

PTH secretion from dispersed bovine parathyroid cells maintained in culture becomes progressively less responsive to changes in the extracellular Ca2+ concentration (Ca2+o) over several days. We have now investigated whether this change in secretory control is associated with alterations in the expression of the Ca2+o-sensing receptor (BoPCaR) recently cloned from bovine parathyroid, which plays a central role in Ca2+o-regulated PTH secretion. BoPCaR messenger RNA levels dropped rapidly in cultured bovine parathyroid cells, as assessed by Northern analysis, decreasing by 78% within 18 h and remaining low for at least 4 days. The level of receptor protein decreased to a comparable extent (approximately 72-82%) after 3-4 days in culture, as determined by immunocytochemistry with specific antibodies directed at the extracellular domain of the receptor. The half-time for the reduction in receptor protein (approximately 2 days) was considerably longer, however, than that for BoPCaR messenger RNA, but was comparable to that for the loss of sensitivity of PTH secretion to Ca2+o. Indeed, there was a close linear correlation between maximal suppressibility of PTH secretion and the intensity of staining for the receptor protein (r = 0.88; P = 0.004). We conclude that alterations in the expression of BoPCaR could explain much of the reduced responsiveness of cultured bovine parathyroid cells to Ca2+o.

Intracellular Ca(2+)-activated K+ channels modulated by variations in extracellular Ca2+ in dispersed bovine parathyroid cells

The modulation of K+ channels by Ca2+ may have important functional implications in parathyroid cells, since in most endocrine cells they control membrane voltage regulating Ca2+ influx and hormone secretion. To characterize specific channel mechanisms regulating membrane voltage in parathyroid cells, the patch-clamp technique was used to determine the activities of K+ channels at different levels of intracellular Ca2+ concentration (Ca2+i) associated with changes in extracellular Ca2+ concentration (Ca2+o). This study shows that the membranes of dispersed bovine parathyroid cells contain a K+ channel that is activated by elevated Ca2+o through an indirect mechanism (i.e. exposure of the entire cell to high Ca2+o activates the channel despite a low Ca2+ concentration within the pipette solution on the external side of the channel under study). This K+ channel has a unitary conductance of 191 pS and is highly selective for K+, similar to the so-called maxi type of Ca(2+)-activated K+ channel previously defined in a number of other cell types. Like the latter channel, the activity of this channel in excised patches from parathyroid cells is markedly increased when an EGTA-containing buffer on the cytoplasmic face of the membrane is replaced with one containing 0.5 microM Ca2+. Changes in Ca2+ on the intracellular side of the membrane also shift the level of voltage necessary for half-maximal activation of the channel from 103 mV at 0.1 microM Ca2+ to 79 mV and 54 mV at 0.25 and 0.5 microM Ca2+, respectively. When similar studies were carried out using cell-attached patches on parathyroid cells exposed to 0.5, 1.5, or 2.0 mM Ca2+o, the values for half-maximal activation were approximately 105, 56, and 29 mV, respectively. The latter result suggests that in intact parathyroid cells, the channel is exposed to Ca2+i concentrations of about 0.15-0.2, 0.4 and 0.6-0.7 microM at these three extracellular Ca2+ concentrations, values that are in excellent agreement with those previously measured using Ca(2+)-sensitive fluorescent dyes. Thus, parathyroid cells express a maxi type of Ca(2+)-activated K+ channel that is indirectly regulated by Ca2+o, presumably through concomitant changes in Ca2+i. The latter may limit the extent of the cellular depolarization produced in response to elevated Ca2+o in this cell type.

Intracellular Ca(2+)-activated K+ channels modulated by variations in extracellular Ca2+ in dispersed bovine parathyroid cells.

The modulation of K+ channels by Ca2+ may have important functional implications in parathyroid cells, since in most endocrine cells they control membrane voltage regulating Ca2+ influx and hormone secretion. To characterize specific channel mechanisms regulating membrane voltage in parathyroid cells, the patch-clamp technique was used to determine the activities of K+ channels at different levels of intracellular Ca2+ concentration (Ca2+i) associated with changes in extracellular Ca2+ concentration (Ca2+o). This study shows that the membranes of dispersed bovine parathyroid cells contain a K+ channel that is activated by elevated Ca2+o through an indirect mechanism (i.e. exposure of the entire cell to high Ca2+o activates the channel despite a low Ca2+ concentration within the pipette solution on the external side of the channel under study). This K+ channel has a unitary conductance of 191 pS and is highly selective for K+, similar to the so-called maxi type of Ca(2+)-activated K+ channel previously defined in a number of other cell types. Like the latter channel, the activity of this channel in excised patches from parathyroid cells is markedly increased when an EGTA-containing buffer on the cytoplasmic face of the membrane is replaced with one containing 0.5 microM Ca2+. Changes in Ca2+ on the intracellular side of the membrane also shift the level of voltage necessary for half-maximal activation of the channel from 103 mV at 0.1 microM Ca2+ to 79 mV and 54 mV at 0.25 and 0.5 microM Ca2+, respectively. When similar studies were carried out using cell-attached patches on parathyroid cells exposed to 0.5, 1.5, or 2.0 mM Ca2+o, the values for half-maximal activation were approximately 105, 56, and 29 mV, respectively. The latter result suggests that in intact parathyroid cells, the channel is exposed to Ca2+i concentrations of about 0.15-0.2, 0.4 and 0.6-0.7 microM at these three extracellular Ca2+ concentrations, values that are in excellent agreement with those previously measured using Ca(2+)-sensitive fluorescent dyes. Thus, parathyroid cells express a maxi type of Ca(2+)-activated K+ channel that is indirectly regulated by Ca2+o, presumably through concomitant changes in Ca2+i. The latter may limit the extent of the cellular depolarization produced in response to elevated Ca2+o in this cell type.

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells.

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.

Regulation of parathyroid hormone messenger RNA levels by protein kinase A and C in bovine parathyroid cells.

Secretion of parathyroid hormone (PTH) is regulated by Ca2+ as well as by protein kinases A and C. In this study we report that protein kinases A and C regulate PTH messenger RNA levels in vitro in dispersed bovine parathyroid cells. Incubation of bovine parathyroid cells with cholera toxin (10(-9) M), which activates adenylate cyclase and indirectly stimulates protein kinase A, increased PTH mRNA levels about 2-fold after 3 and 7 h incubation, but not at 24 h. Incubation with pertussis toxin (5 x 10(-9) M), which blocks the high-calcium-mediated inhibition of cyclic adenosine monophosphate accumulation in these cells, also reversed the inhibition of PTH mRNA levels at high Ca2+ (2.0 mM) with a marked increase in PTH mRNA levels. Pertussis toxin also increased PTH mRNA at a low extracellular Ca2+ concentration (0.7 mM) (4-fold increase) and a normal concentration (1.25 mM) (2-fold increase). Inhibition of protein kinase C both by staurosporine (1 x 10(-8) M) and by prolonged incubation with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (1 x 10(-7) M), decreased PTH mRNA levels at 24 h, reaching approximately 40% and 5% of control, respectively. Staurosporine and PMA had no effect on PTH mRNA levels at 3 h. The inactive phorbol ester, phorbol 12-13-dibutyrate (PDBu), had no effect on PTH mRNA levels at 1 and 24 h. There were no changes in a control gene 18S RNA in these studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone (PTH) secretion: stimulation of PTH secretion by a peptide derived from the adenosine diphosphate-ribosylation factor

Using preparations of dispersed bovine parathyroid cells, we have investigated the effect of a 16-residue synthetic peptide, ARF-16, which corresponds to the N-terminus of the ADP-ribosylation factor, on the secretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrations in the range of 15-25 microM, the ARF peptide stimulated PTH secretion to a greater degree than low extracellular calcium, and at 25 microM was more effective than isoproterenol. The stimulatory effect of ARF was not dependent on the extracellular calcium concentration over the range of 0.5-3 mM. Upon testing other synthetic peptides of similar size we found no effect on PTH secretion, indicating that the ARF-16 effect is specific. In an attempt to define the structural elements of ARF that are required for activity, we tested several analogs of ARF with amino acids deleted from the N- and C-terminus. Deletion of the 2 N-terminal residues yielded a peptide with substantially reduced activity. Further deletions from the N-terminus yielded an inactive peptide. Similarly, a peptide with deletions of 3 residues from the C-terminus was inactive. Thus, the activity of ARF-16 requires both the N- and C-terminal sequence, suggesting that the 16-residue peptide is the minimal sequence required for full activity. Measurements of cAMP concentrations indicate that the stimulatory effect is not mediated via this second messenger. The ARF peptide does not alter intracellular calcium, suggesting that its effect is not mediated by calcium. Although cells incubated with ARF are vigorously stimulated to secrete PTH, this effect is reversible, as demonstrated by washing cells free of ARF, whereupon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion through a low affinity interaction at the cell surface. Other experiments, in which the capacity for ARF stimulation was abolished after a brief exposure of the cells to trypsin, support this conclusion. Characteristics of the ARF stimulatory effect, such as dose dependency and reversibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on extracellular calcium levels and is not mediated via cAMP, we believe that this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.

Parathyroid hormone (PTH) secretion: stimulation of PTH secretion by a peptide derived from the adenosine diphosphate-ribosylation factor.

Using preparations of dispersed bovine parathyroid cells, we have investigated the effect of a 16-residue synthetic peptide, ARF-16, which corresponds to the N-terminus of the ADP-ribosylation factor, on the secretion of PTH. We find it to be a very effective secretagogue for PTH secretion, acting in a dose- and time-dependent manner. At concentrations in the range of 15-25 microM, the ARF peptide stimulated PTH secretion to a greater degree than low extracellular calcium, and at 25 microM was more effective than isoproterenol. The stimulatory effect of ARF was not dependent on the extracellular calcium concentration over the range of 0.5-3 mM. Upon testing other synthetic peptides of similar size we found no effect on PTH secretion, indicating that the ARF-16 effect is specific. In an attempt to define the structural elements of ARF that are required for activity, we tested several analogs of ARF with amino acids deleted from the N- and C-terminus. Deletion of the 2 N-terminal residues yielded a peptide with substantially reduced activity. Further deletions from the N-terminus yielded an inactive peptide. Similarly, a peptide with deletions of 3 residues from the C-terminus was inactive. Thus, the activity of ARF-16 requires both the N- and C-terminal sequence, suggesting that the 16-residue peptide is the minimal sequence required for full activity. Measurements of cAMP concentrations indicate that the stimulatory effect is not mediated via this second messenger. The ARF peptide does not alter intracellular calcium, suggesting that its effect is not mediated by calcium. Although cells incubated with ARF are vigorously stimulated to secrete PTH, this effect is reversible, as demonstrated by washing cells free of ARF, whereupon PTH secretion returns to basal levels. These results indicate that the peptide is not entering the cells, but is effecting secretion through a low affinity interaction at the cell surface. Other experiments, in which the capacity for ARF stimulation was abolished after a brief exposure of the cells to trypsin, support this conclusion. Characteristics of the ARF stimulatory effect, such as dose dependency and reversibility, lead us to conclude that the peptide is probably acting on the regulated secretory pathway. As the effect is not dependent on extracellular calcium levels and is not mediated via cAMP, we believe that this peptide will be a useful additional tool for future studies of the mechanisms of PTH secretion.

Effects of high extracellular calcium concentrations on phosphoinositide turnover and inositol phosphate metabolism in dispersed bovine parathyroid cells.

We previously showed that high extracellular calcium (Ca2+) concentrations raise the levels of inositol phosphates in bovine parathyroid cells, presumably via the G protein-coupled, "receptor-like" mechanism through which Ca2+ is thought to regulate these cells. To date, however, there are limited data showing Ca(2+)-evoked hydrolysis of phosphoinositides with attendant increases in the levels of the biologically active 1,4,5 isomer of inositol trisphosphate (IP3) that would be predicted to arise from such a receptor-mediated process. In the present studies we used HPLC and TLC, respectively, to quantify the high Ca(2+)-induced changes in various inositol phosphates, including the isomers of IP3, and phosphoinositides in bovine parathyroid cells prelabeled with [3H]inositol. In the absence of lithium, high Ca2+ dose dependently elevated the levels of inositol-1,4,5-trisphosphate [I(1,4,5)P3], with a maximal, 4- to 5-fold increase within 5 s; the levels of inositol 1,3,4-trisphosphate [I(1,3,4)P3] first rose significantly at 5-10 s and remained 5- to 10-fold elevated for at least 30 minutes. These changes were accompanied by reciprocal 29-36% decreases in PIP2 (within 5-10 s, the earliest time points examined), PIP (within 60 s), and PI (within 60 s). These results document that, as in other cells responding to more classic "Ca(2+)-mobilizing" hormones, the high Ca(2+)-evoked increases in inositol phosphates in bovine parathyroid cells arise from the hydrolysis of phosphoinositides, leading to the rapid accumulation of the active isomer of IP3. The latter presumably underlies the concomitant spike in the cytosolic calcium concentration (Ca(i)) in parathyroid cells.

Structure-function relationships for the effects of various aminoglycoside antibiotics on dispersed bovine parathyroid cells

Structure-function relationships for the effects of various aminoglycoside antibiotics on dispersed bovine parathyroid cells

A comparison of the effects of concanavalin-A and tetradecanoylphorbol acetate on the modulation of parathyroid function by extracellular calcium and neomycin in dispersed bovine parathyroid cells

Ca2+ and other polyvalent cations as well as polycations, such as neomycin, produce similar effects on intracellular second messengers and PTH release in dispersed bovine parathyroid cells, but it is unclear whether all of these agents share the same mechanism of action. The lectin Concanavalin-A (Con-A) and the activator of protein kinase-C tetradecanoylphorbol acetate (TPA) blunt the effects of elevated extracellular calcium (Ca2+) concentrations on several aspects of parathyroid function, including PTH release, the cytosolic calcium concentration, and the accumulation of cAMP and inositol phosphates. In the present studies we used these two agents as well as pertussis toxin as probes to investigate further whether neomycin acts on parathyroid cells through the same receptor-like mechanism used by extracellular Ca2+ to regulate parathyroid function. Con-A and TPA both enhanced PTH release by about 2-fold at 0.5-1 x 10(-4) M neomycin, concentrations that inhibited PTH release to an extent (40-50%) similar to that seen with high (1.5-2 mM) Ca2+. Con-A also reduced the inhibition of agonist-stimulated cAMP accumulation by the same concentrations of neomycin. Conversely, Con-A and TPA produced 70-80% decreases in the cytosolic calcium concentration transient and the accumulation of inositol phosphates stimulated by neomycin. The effects of these two agents on neomycin-regulated parathyroid function were similar in magnitude to their actions on the modulation of these same parameters by extracellular Ca2+. Pertussis toxin, however, which we have previously shown to block the inhibitory effects of high Ca2+ and neomycin on cAMP accumulation, had no effect on the inhibition of PTH release by these two agents. These results provide further indirect evidence that polyvalent cations and polycations act on the parathyroid cell through related pathways, which probably involve cell surface moieties containing carbohydrate(s).