Bovine Parathyroid Cells Research Articles

Parathyroid hormone mRNA levels are increased by progestins and vary during rat estrous cycle.

Estrogen increases parathyroid hormone (PTH) mRNA levels in vivo in ovariectomized rats. We now show that the 19-norprogestin R-5020 given to weanling rats or mature ovariectomized rats led to a twofold increase in thyroparathyroid PTH mRNA levels. This increase in PTH mRNA occurred at 24 and 48 h after progesterone but not at 72 h. There were no changes in serum calcium. In vitro, in primary cultures of bovine parathyroid cells, progesterone increased PTH mRNA levels threefold at 10(-8) M and twofold at 10(-9) M after 24 h. Progesterone receptor (PR) mRNA was demonstrated in rat parathyroid tissue by in situ hybridization and in human parathyroid adenoma by immunohisto-chemistry. Changes in PTH mRNA levels during the rat estrous cycle were also studied. At proestrus and estrus PTH mRNA levels were increased significantly by three- and fourfold compared with diestrus. Our results confirm that the parathyroid gland is a target organ for the ovarian sex steroids estrogen and progesterone and are of physiological relevance as shown by the changes during estrus.

Investigation of calcium-induced hydrolysis of phosphoinositides in normal and lithium-treated parathyroid cells

Lithium-induced hyperparathyroidism is characterized by a reduction in parathyroid sensitivity to changes in extracellular calcium (Ca2+). Ca(2+)-induced transmembrane signal transduction in the parathyroid cell is known to result in the hydrolysis of phosphatidylinositol bisphosphate (PIP2), generating increases in intracellular inositol phosphates, a process which is mediated by a calcium receptor. To determine if lithium's effect on parathyroid cell function is mediated by an alteration in Ca(2+)-induced hydrolysis of PIP2, inositol 4-monophosphate (IP1), and inositol 1,4,5-trisphosphate (IP3) were measured using anion-exchange chromatography in normal and lithium chloride (LiCl)-treated bovine parathyroid cells at Ca2+ concentrations varying from 0.5 mmol/L to 5.0 mmol/L. IP1 and IP3 concentrations were determined in terms of percent control, defined as the IP1 or IP3 concentration at an [Ca2+] of 0.5 mmol/L. Increases in [IP1]/10(6) cells (mean +/- standard error of the mean [SEM]) in response to progressive increases in Ca2+ from 0.5 mmol/L to 5.0 mmol/L varied from 825 +/- 228 to 4,474 +/- 382 in control cells versus 1,139 +/- 243 to 4,689 +/- 630 in cells pretreated with LiCl (P > 0.05). The increases in [IP3]/10(6) cells (mean +/- SEM) in response to increases in Ca2+ from 0.5 mmol/L to 5.0 mmol/L, varied from 146 +/- 14 to 385 +/- 35 in control cells versus 134 +/- 16 to 327 +/- 55 in cells pretreated with LiCl (P > 0.05). Our results demonstrate that LiCl does not effect Ca(2+)-induced hydrolysis of PIP2, suggesting that the desensitizing effect of LiCl on the parathyroid cell is not the result of a Ca2+ receptor-mediated phenomenon.

Suppression of PTH secretion by 1α,25-dihydroxy-dihydotachysterol in isolated bovine parathyroid cells in tissue culture

Suppression of PTH secretion by 1α,25-dihydroxy-dihydotachysterol in isolated bovine parathyroid cells in tissue culture

Calcium-activated proteases in the bovine parathyroid gland: potential role in degradation of parathyroid hormone to peptide fragments.

Our studies suggest that protein kinase C is involved in low calcium (Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not directly in high Ca(2+)-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca(2+)-regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either micro-calpain or Ca2+, to 71.5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47-48 and bPTH69-84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca(2+)-dependent degradation of PTH to secreted C-terminal fragments.

Loss of Calcium Responsiveness in Cultured Bovine Parathyroid Cells Is Associated with Decreased Calcium Receptor Expression

Suppression of PTH secretion by extracellular calcium is mediated by a plasma membrane calcium receptor (CaR). However, primary cultures of bovine parathyroid cells are known to quickly lose their responsiveness to extracellular calcium. The present study was designed to determine if the loss of calcium responsiveness is due to changes in CaR expression. In primary monolayer cultures of parathyroid cells, calcium-mediated suppression of PTH was still evident after 24 hours in culture but was completely absent after 6 days. This was preceded by a 75% drop in CaR mRNA content within 24 hours. CaR mRNA levels remained low for the 6-day culture. Earlier time points, examined in parathyroid cell suspensions, showed a 70% drop in CaR mRNA by 4 hours after collagenase-dispersion of the glands and an 85% drop after 24 hours. The decreased expression of CaR mRNA was not influenced by altering medium serum, calcium, or 1,25-dihydroxyvitamin D3. Our results indicate that the loss of responsiveness of cultured parathyroid cells to calcium is due to decreased CaR mRNA and, presumably, CaR protein expression.

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.

Spontaneous oscillations of intracellular calcium in single bovine parathyroid cells may be associated with the inhibition of parathyroid hormone secretion.

PTH secretion is tightly regulated by extracellular calcium ([Ca2+]e) and in turn regulates calcium homeostasis through its action on target tissues. We investigated the mechanism and physiological significance of intracellular calcium ([Ca2+]i) levels in relation to the secretion of PTH in single bovine parathyroid cells. [Ca2+]i was recorded using digital imaging microscopy, and secretion of PTH was correlated in the same cell using the reverse hemolytic plaque assay. In individual parathyroid cells, oscillations of [Ca2+]i were present in response to specific stimuli. Like secretory activity, response to [Ca2+]e concentrations was heterogeneous. Oscillations of [Ca2+]i occurred spontaneously in 22% of cells at inhibitory concentrations of [Ca2+]e. Oscillations were present only in high [Ca2+]e (> or = 1.8 mM) and not noted at lower concentrations of [Ca2+]e. The interval and amplitude of [Ca2+]i oscillations were 42 +/- 2 sec, and 20 +/- 1 nM (mean +/- SE), respectively. Oscillations were rapidly abolished when [Ca2+]e was removed by EGTA, and this effect was reversible. Addition of Mg2+ or polycationic antibiotics such as neomycin resulted in an [Ca2+]i spike, but oscillations were absent. Lanthanum, which blocks Ca2+ influx through calcium channels in various cells, rarely caused oscillations even in the presence of high concentrations of [Ca2+]e. To test the role of cAMP in [Ca2+]i oscillations, we added the beta-agonist isoproterenol. The addition of isoproterenol, however, did not cause oscillations. The number of cells that released PTH was significantly lower in cells with oscillations compared with cells without oscillations. We suggest that spontaneous [Ca2+]i oscillations are due to the influx of [Ca2+]e through ion channels rather than release from [Ca2+]i stores and may be a specific intracellular signal associated with inhibition of PTH secretion.

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

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

Spontaneous oscillations of intracellular calcium in single bovine parathyroid cells may be associated with the inhibition of parathyroid hormone secretion

PTH secretion is tightly regulated by extracellular calcium ([Ca2+]e) and in turn regulates calcium homeostasis through its action on target tissues. We investigated the mechanism and physiological significance of intracellular calcium ([Ca2+]i) levels in relation to the secretion of PTH in single bovine parathyroid cells. [Ca2+]i was recorded using digital imaging microscopy, and secretion of PTH was correlated in the same cell using the reverse hemolytic plaque assay. In individual parathyroid cells, oscillations of [Ca2+]i were present in response to specific stimuli. Like secretory activity, response to [Ca2+]e concentrations was heterogeneous. Oscillations of [Ca2+]i occurred spontaneously in 22% of cells at inhibitory concentrations of [Ca2+]e. Oscillations were present only in high [Ca2+]e (> or = 1.8 mM) and not noted at lower concentrations of [Ca2+]e. The interval and amplitude of [Ca2+]i oscillations were 42 +/- 2 sec, and 20 +/- 1 nM (mean +/- SE), respectively. Oscillations were rapidly abolished when [Ca2+]e was removed by EGTA, and this effect was reversible. Addition of Mg2+ or polycationic antibiotics such as neomycin resulted in an [Ca2+]i spike, but oscillations were absent. Lanthanum, which blocks Ca2+ influx through calcium channels in various cells, rarely caused oscillations even in the presence of high concentrations of [Ca2+]e. To test the role of cAMP in [Ca2+]i oscillations, we added the beta-agonist isoproterenol. The addition of isoproterenol, however, did not cause oscillations. The number of cells that released PTH was significantly lower in cells with oscillations compared with cells without oscillations. We suggest that spontaneous [Ca2+]i oscillations are due to the influx of [Ca2+]e through ion channels rather than release from [Ca2+]i stores and may be a specific intracellular signal associated with inhibition of PTH secretion.

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.

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.

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)

Lithium-induced alterations in parathyroid cell function: Insight into the pathogenesis of lithium-associated hyperparathyroidism

Reduced parathyroid sensitivity to changes in calcium (Ca2+) has been observed in patients treated with lithium (Li+). In order to investigate this desensitization phenomenon, the effect of Li+ on cytosolic calcium (Cai2+) regulation was examined. Transmembrane signal transduction and Ca2+ sequestration were investigated in bovine parathyroid cells by measuring changes in [Cai2+] in response to 5 mmol/L magnesium (Mg2+), 0.5 to 2.5 mmol/L Ca2+, 25 mumol/L adenosine triphosphate (ATP), and 1 mumol/L ionomycin in cells pretreated with 1 to 10 mmol/L lithium chloride (LiCl) and control cells. Measurement of Cai2+ was made using fura-2. Increases in [Cai2+] in response to Ca2+ and Mg2+ were blunted following overnight culture with as low as 1 mmol/L LiCl. In normocalcemic medium, 1 mmol/L Ca2+ produced an 81% increase in [Cai2+] in control cells compared with a 58% increase in cells pretreated with LiCl (P < 0.01), whereas in hypocalcemic medium, increases in [Cai2+] were similar in lithium-treated and control cells (78% versus 82%, P > 0.1). The ATP produced increases in [Cai2+] from 225 +/- 9 nmol/L to 366 +/- 10 nmol/L in control cells, compared with 221 +/- 7 nmol/L to 308 +/- 10 nmol/L in cells pretreated with 5 mmol/L LiCl (P < 0.01). Ionomycin-induced increases in [Cai2+] were unaffected by Li+. We concluded that the in vitro desensitizing effects of Li+ occur at therapeutic concentrations, but only in the presence of Ca2+ in concentrations that induce transmembrane signaling; and that Li+ blunts increases in [Cai2+] related to cation and ATP-induced transmembrane signal transduction without affecting ionomycin-releasable Ca2+ stores.

Stimulus-secretion coupling in parathyroid cells deficient in protein kinase C activity

The role of protein kinase C (PKC) in regulating cytosolic Ca2+ concentrations ([Ca2+]i) and parathyroid hormone (PTH) secretion was studied in bovine parathyroid cells rendered deficient in PKC activity by incubation with phorbol 12-myristate 13-acetate (PMA). Pretreatment with PMA caused a time- and concentration-dependent loss of functional PKC activity as assessed by the failure of [Ca2+]i and PTH secretion to respond to the subsequent addition of PKC activators or the inhibitor staurosporine. Pretreatment for 24 h with 1 microM PMA caused a loss of 85% of the total and 98% of the cytosolic PKC activity. Cells so pretreated were considered to be "PKC downregulated." Increasing the concentration of extracellular Ca2+ or Mg2+ caused corresponding increases in [Ca2+]i that were similar in control and in PKC-downregulated cells. PTH secretion regulated by extracellular Ca2+ or Mg2+ was likewise similar in control and PKC-downregulated cells. Stimulus-secretion coupling is thus unimpaired in parathyroid cells deficient in PKC activity. Cytosolic Ca2+ responses remained depressed in cells incubated for 24 h with low concentrations of PMA (30 or 100 nM). However, under these conditions, extracellular Ca2+ still suppressed PTH secretion similarly to control cells. These results reveal a dissociation between cytosolic Ca2+ and PTH secretion and suggest that signals other than cytosolic Ca2+ are involved in the regulation 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.

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.

Paracrine interactions among parathyroid cells: effect of cell density on cell secretion.

Cell-cell interactions are important in the regulation of endocrine cell secretion. To investigate the possibility that cell communication may alter the regulation of parathyroid cell secretion, we utilized the reverse hemolytic plaque assay (RHPA) to measure parathyroid hormone (PTH) release from individual cells. Bovine parathyroid cells were dispersed and plated with protein A-conjugated erythrocytes at cell densities ranging from 0.9 to 36 x 10(2) cells/cm2 in 0.2 mM calcium. Cell populations were greater than 98% homogenous as determined by immunocytochemistry and in situ hybridization for PTH mRNA. Plaques were developed and data analyzed for the amount of PTH per cell released (plaque area in microns 2 x 10(4)) and the determination of cell recruitment (% plaques formed). A positive correlation existed between parathyroid cell density and the amount of PTH released. As the distance between cells increased, the plaque area (amount of PTH released per cell) decreased (ranging from 1.0 x 10(4) microns 2 at 0.9 x 10(2) cells/cm2 versus 1.6 x 10(4) microns 2 at 36 x 10(2) cells/cm2). The percentage of cells releasing PTH (recruitment) also decreased (16% at 0.9 x 10(2) cells/cm2 versus 47% at 36 x 10(2) cells/cm2). These data suggest that parathyroid cells in close proximity are stimulated to secrete more hormone than those at lesser densities. In addition, parathyroid cells are recruited to secrete PTH when plated at high density. Factor(s) released by the parathyroid cell may increase cell responsiveness and stimulate secretion in a paracrine fashion.

The release of parathyroid hormone and the exocytosis of a proteoglycan are modulated by extracellular Ca2+ in a similar manner.

Secretion of parathyroid hormone (PTH) is regulated in part by a classical "stimulus-secretion" pathway responsive to catecholamines. The primary physiological modulator of PTH exocytosis in parathyroid cells, however, is extracellular free Ca2+. Ca(2+)-modulated PTH release exhibits several characteristics suggestive of constitutive secretion. The aim of this work was to obtain further information about the possible intracellular origins of Ca(2+)-modulated exocytosis in parathyroid cells. Freshly dissociated bovine parathyroid cells labeled with [35S]sulfate synthesized a soluble chondroitin/dermatan sulfate proteoglycan (M(r) approximately 90-150 K) that was secreted into the medium. The export of [35S]sulfated proteoglycan satisfied several criteria that generally define constitutive release: 1) export is detected in the medium shortly (7-15 min) after a 5-min pulse, 2) there is minimal intracellular storage after equilibrium labeling (because of combined processes of rapid release and intracellular degradation), and 3) there is insensitivity to stimulation with isoproterenol, a known secretagogue in parathyroid cells. Nevertheless, the increase in extracellular Ca2+ from 0.5 to 2.0 mM reduced the export of the [35S]sulfated proteoglycan from 60% of total labeled to 30%. In addition, a secreted pool of immunoreactive PTH and [35S]sulfated proteoglycan was modulated by external Ca2+ to the same degree and sensitivity, although isoproterenol was more effective in stimulating the release of PTH than that of proteoglycan. Together, our experimental results show that in the parathyroid cell extracellular Ca2+ modulates negatively the export of both PTH and proteoglycan, a putative marker for constitutive secretion. We further suggest that a portion of newly synthesized PTH also enters this pathway, whereas another portion proceeds to an isoproterenol-releasable compartment from which the proteoglycan is largely excluded.

Inhibition of parathyroid hormone secretion by amino-terminal chromogranin peptides.

The parathyroid gland responds to decreases in levels of extracellular calcium by increasing the secretion of both PTH and chromogranin-A (CGA) in approximately equal molar ratios. Because CGA has been suggested to be a precursor for biologically active peptides, we used primary cultures of bovine parathyroid cells to examine the effects of various peptides from CGA as well as analogous peptides from chromogranin-B (CGB) on PTH secretion. In concentrations from 10(-8)-10(-7) M, amino-terminal peptide CGA-(1-76) effectively inhibited the release of PTH in response to low extracellular calcium. Truncated analogs of this peptide, CGA-(1-40), CGB-(1-41), and CGA-(17-38) were also found to be active in the following order: CGA-(1-76) = CGA-(1-40) = CGB-(1-41) > CGA-(17-38). The biological activity of CGA-(1-40) was markedly reduced after reduction and alkylation, which resulted in disruption of the single disulfide bond between Cys17 and Cys38. Moreover, peptides derived from other regions of CGA and CGB, which included CGA-(403-428), CGB-(1-16), CGB-(316-326), and CGB-(635-657) were inactive. Pulse-chase experiments, using primary cultures of bovine parathyroid cells, revealed the presence of a CGA peptide in the culture medium that had the same amino-terminal sequence and mobility on sodium dodecyl sulfate-polyacrylamide gels as synthetic CGA-(1-76). Furthermore, in binding and cross-linking studies using intact parathyroid cells, CGA-(1-40) formed a single, covalently linked protein complex with a mol wt of 78,000. Formation of the protein complex could be completely inhibited in the presence of an excess of either CGB-(1-41) or CGA-(17-38). These results show that a naturally occurring amino-terminal peptide from CGA as well as shorter analogs can act as potent inhibitors of PTH secretion, and that their biological activity may be mediated through binding to a specific cell surface protein.