Regulation of parathyroid hormone secretion.

Calcium is the most important physiological regulator of PTH secretion. Peak PTH secretion occurs at an intracellular calcium concentration of about 200 nM, regardless of the extracellular calcium concentration. We suggest, therefore, that intracellular calcium concentration is a regulator of PTH secretion that maintains calcium homeostasis. Other factors may be responsible for modulation of the intracellular calcium concentration, ultimately modulating PTH secretion. The "paradoxical" nature of the dependence of PTH secretion on the calcium concentration may be explained by considering PTH secretion to be unusual in a quantitative, rather than a qualitative, fashion. A possible mechanism for the control of PTH secretion by intracellular calcium, which involves calcium-activated potassium channels, is proposed. The parathyroid cell plasma membrane contains several sensors or channels by means of which the cell senses extracellular calcium. It is not clear whether these entities are coupled to each other or whether they function independently. Guanine nucleotide regulatory proteins are transducers of extracellular signals, including calcium. Several other second messengers that influence PTH secretion have also been described, but possible interactions between these messengers have not yet been determined.

The biphasic calcium dose-response curve for parathyroid hormone secretion in electropermeabilized adult bovine parathyroid cells.

We have used the method of electropermeabilization to measure the dependence of PTH secretion on internal calcium concentration in adult bovine parathyroid cells. The dose-response curve is biphasic, with a peak at 10(-7) M calcium. This result differs significantly from the dose-response curves that have been determined by this method for many other secretory systems, where secretion requires much more calcium and the dependence of secretion on calcium is monotonic. Guanine nucleotide analogs did not modify the calcium dose-response curve of PTH secretion in the electropermeabilized parathyroid cells. The unique properties of adult parathyroid cell secretion are analogous to the unique properties of parathyroid calcium-activated potassium channels, which differ from calcium-activated potassium channels of other cells in that they open at unusually low calcium concentrations and have a peak open probability at about 10(-7) M calcium. We suggest that the opening of these channels in secretory vesicles is required for secretion.

Role of calcium channels in parathyroid hormone secretion.

Experimental evidence exists for the presence of parathyroid cell membrane calcium channels that respond to plasma calcium. In previous reports, the effects of various calcium channel agents on PTH secretion have revealed conflicting results. To resolve some of these inconsistencies, we have compared the pure calcium channel agonist, (+)202-791, and its antagonistic enantiomer (-)202-791 with other calcium channel agents--verapamil, nifedipine, and (+)Bay-K-8644. The agonist (+)202-791 enhanced 45Ca+2 uptake and decreased PTH secretion, while the antagonist (-)202-791 decreased 45Ca+2 uptake and increased PTH secretion. The calcium channel appears coupled to a G-protein as indicated by pertussis toxin treatment of the cells. The enantiomers (+/-)202-791 had little effect on intracellular cAMP production suggesting that the calcium channel may not be responsible for the previously observed calcium-mediated changes in cAMP. The antagonist (-)202-791 increased the phosphorylation of a 60-kd protein. The enantiomers (+/-)202-791 did not alter the effect of depolarizing concentrations of potassium on PTH secretion. Our results suggest that calcium channels provide a pathway for the movement of calcium across the plasma membrane and that this pool of calcium regulates, at least in part, PTH secretion.

Adenosine 3',5'-cyclic monophosphate-mediated enhancement of calcium-evoked prolactin release from electrically permeabilised 7315c tumour cells.

1. The 7315c tumour cell was used as a model system for the investigation of adenosine 3',5'-cyclic monophosphate (cyclic AMP)-mediated enhancement of calcium-evoked prolactin release. 2. 7315c cells were permeabilised by subjecting the cells to intense electric fields. Studies investigating the penetration of the dye ethidium bromide indicated that the cells were completely permeabilised after 2 discharges of 2000 volts and that the pores remained open for at least 30 min before beginning to reseal. These permeabilisation parameters were consistent with those which gave maximal calcium-stimulated prolactin release. 3. In the absence of calcium and in the presence of EGTA (1 mM), permeabilised 7315c cells secreted prolactin at a rate of 0.23 ng min-1 per 10(6) cells. When EGTA was replaced by 1.5 mM calcium, permeabilised cells secreted prolactin at a rate of 2.20 +/- 0.30 ng min-1 per 10(6) cells in the first 5 min of exposure. Maximal calcium-dependent prolactin secretion from permeabilised cells occurred at 37 degrees C. 4. The amount of prolactin secreted, in a 5 min incubation at 37 degrees C, from permeabilised cells depended upon the free calcium concentration in the permeabilisation medium. Calcium stimulated prolactin release from permeabilised cells in the concentration range 0.1-10 microM (half maximal = 5.8 microM). When permeabilised cells were exposed to cyclic AMP (100 microM) for 5 min prior to and during a 5 min challenge with various concentrations of calcium, the amount of prolactin secreted at each effective concentration of calcium was increased. However, cyclic AMP did not alter the potency of calcium as a stimulant of prolactin secretion. 5. The results suggest that cyclic AMP potentiates calcium-evoked secretion from 7315c cells, not by increasing the entry of calcium into the cytosol, but at a step in the secretory process, distal to calcium entry, which modulates the ability of an increase in cytosolic calcium concentration to stimulate prolactin release.

Evaluation of a hyperbaric system to be used in conjunction with a fluorometer.

A high-pressure chamber that can be used inside the sample chamber of a spectrofluorometer is described and some performance characteristics are presented. The chamber body, constructed of 316 stainless steel, is temperature regulated using resistive heating elements and a microprocessor-based proportional integral derivative controller. The chamber holds a standard 1-cm2 cuvette that indexes with an electromagnetic stirrer. Injection of different solutions into the closed and pressurized (6.8 MPa) vessel is accomplished by computer-controlled, low-volume solenoids attached to separate microliter injection ports. Repetitive injections of fluids down to a volume of 7 microliters are possible in the pressurized chamber. Temperature stability of the chamber is +/- 0.2 degrees C at atmospheric or elevated pressure. However, during the initial phase (first 3 min) of pressurization, at a compression rate of 0.62 MPa/min, a 0.23 degrees C/min increase in temperature occurs. The chamber windows depress the relative intensity of the emitted light by approximately 20% for visible light and 40% for near UV; however, total sensitivity of the system is sufficient to accomplish most determinations while maintaining a good signal-to-noise ratio. This system can be used to evaluate the response of several molecular and cellular events during compression and at depth with the use of various fluorometric probes.

Effects of phorbol ester on tyrosine hydroxylase phosphorylation and activation in cultured bovine adrenal chromaffin cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused phosphorylation of phosphoproteins of 56-kDa which co-migrated with and had identical pI values to subunits of tyrosine hydroxylase. The phosphorylation was closely correlated with an increase of [3H]3,4-dihydroxyphenylalanine (DOPA) production which is a reflection of increased tyrosine hydroxylase activity. Only those phorbol esters which activate protein kinase C induced phosphorylation of the 56-kDa proteins and increased [3H]DOPA production. Neither TPA-induced phosphorylation of the 56-kDa proteins nor TPA-induced enhancement of [3H] DOPA production required extracellular Ca2+. TPA caused increases in phosphorylation of the 56-kDa proteins and increases in [3H]DOPA production over similar concentration ranges (10-1000 nM). TPA did not increase cellular cAMP. The data suggest that phorbol ester-induced phosphorylation of intracellular tyrosine hydroxylase, possibly by protein kinase C, results in increased tyrosine hydroxylase activity.

Cholinergic receptor-mediated phosphorylation and activation of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells.

We have identified a 56-kilodalton protein in cultured bovine adrenal chromaffin cells that is phosphorylated when catecholamine secretion is stimulated. Immunodetection on Western blots from both one- and two-dimensional polyacrylamide gels indicated that this protein was tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. Two-dimensional polyacrylamide gel electrophoresis of proteins from unstimulated cells revealed small amounts of phosphorylated protein with a molecular weight of 56K and pI values of 6.37 and 6.27 which were subunits of tyrosine hydroxylase. Nicotinic stimulation of chromaffin cells caused the phosphorylation of three proteins of 56 kilodaltons with pI values of approximately 6.37, 6.27, and 6.15 which were tyrosine hydroxylase. The immunochemical analysis also revealed that there was unphosphorylated tyrosine hydroxylase 56 kilodaltons with a pI of 6.5 which may have decreased on nicotinic stimulation. The phosphorylation of tyrosine hydroxylase was associated with an increase in in situ conversion of [3H]tyrosine to [3H]dihydroxyphenylalanine ([3H]DOPA). Muscarinic stimulation also caused phosphorylation of tyrosine hydroxylase, but to a smaller extent than did nicotinic stimulation. The secretagogues, elevated K+ and Ba2+, stimulated phosphorylation of tyrosine hydroxylase and [3H]DOPA production. The effects of nicotinic stimulation and elevated K+ on tyrosine hydroxylase phosphorylation and [3H]DOPA production were Ca2+-dependent. Nicotinic agonists also raised cyclic AMP levels in chromaffin cells after 2 min. Dibutyryl cyclic AMP and forskolin, which have little effect on catecholamine secretion, also caused phosphorylation of tyrosine hydroxylase. These stimulators of cyclic AMP-dependent processes caused the appearance of two phosphorylated subunits of tyrosine hydroxylase with pI values of 6.37 and 6.27. There was also a small amount of phosphorylated subunit with a pI of 6.15. Both agents stimulated [3H]DOPA production. The experiments indicate that tyrosine hydroxylase is phosphorylated and activated when chromaffin cells are stimulated to secrete. The data suggest that the earliest phosphorylation of tyrosine hydroxylase induced by a nicotinic agonist occurs through stimulation of a Ca2+-dependent protein kinase. After 2 min phosphorylation by a cyclic AMP-dependent protein kinase may also occur. Phosphorylation of tyrosine hydroxylase is associated with an increase in in situ tyrosine hydroxylase activity.

Levels of biogenic amines, their metabolites, and tyrosine hydroxylase activity in the human epileptic temporal cortex.

The levels of serotonin (5-HT), 5 hydroxyindoleacetic acid (5-HIAA), dopamine (DA), homovanillic acid (HVA), norepinephrine (NE), and tyrosine hydroxylase (TH) activity were measured in the focus (spiking) and nonfocus (nonspiking) regions of the temporal neocortex of 20 patients with intractable complex partial seizures. The levels of 5-HT, DA, 5-HIAA, and HVA were higher in the focus when compared to the nonfocus. Values for NE and TH activity were not different when focus and nonfocus were compared. The ratios of metabolite to precursor for 5-HT and DA were not significantly different between the focus and the nonfocus, suggesting that the changes observed were the result of a modification in the synthesis and release of these amines. Such changes in the epileptic focus could be caused by altered transsynaptic regulatory processes, which occur as a result of neuronal loss, gliosis, or neuronal sprouting.

Effects of phorbol ester on catecholamine secretion and protein phosphorylation in adrenal medullary cell cultures.

The effects of phorbol 12-myristate 13-acetate (PMA) on catecholamine secretion and protein phosphorylation from intact and digitonin-treated chromaffin cells were investigated. PMA (10-300 nM), an activator of protein kinase C, caused a slow Ca2+-dependent release of catecholamine from intact chromaffin cells that was potentiated by the Ca2+ ionophore ionomycin. PMA also enhanced secretion induced by Ba2+. In cells with plasma membranes rendered permeable by digitonin to Ca2+, ATP, and protein, PMA (100 nM) enhanced Ca2+-dependent secretion approximately 70% at 0.5 microM Ca2+ and 30% at 10 microM Ca2+. PMA enhanced the maximal response to Ca2+ approximately 25% and decreased the Ca2+ concentration required for half-maximal secretion approximately 30%. The effects of PMA on chromaffin cells were associated with a 2- to 3-fold increase in the phosphorylation of a 56-kDa protein that may be tyrosine hydroxylase. Other proteins were phosphorylated to a lesser extent. The experiments suggest that PMA increases protein kinase activity and secretion in chromaffin cells and raise the possibility that protein kinase C modulates catecholamine secretion in chromaffin cells.