Release of growth hormone from ox pituitary slices after pronase treatment.

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).

The effect of acetylcholine on inositol lipid metabolism and intracellular calcium concentrations in bovine anterior pituitary cells.

Acetylcholine (ACh) increased the intracellular calcium concentration in bovine anterior pituitary cells. In the presence of the calcium channel antagonists verapamil (20 microM) or nitrendepine (1 microM) the increase in calcium was partially inhibited but showed both transient and sustained components. In the presence of EGTA (2.5 mM) only the transient component was observed. ACh also decreased inositol radioactivity in phosphatidylinositides and increased it in inositol phosphates. It is concluded that the increase in calcium caused by acetylcholine requires both the entry of external calcium and mobilisation of internal calcium. Replacement of external sodium by N-methyl-D-glucamine inhibited the rises in calcium and inositol phosphate labelling in response to ACh. Tetrodotoxin (3 microM) or ouabain (50 microM) did not affect either response to ACh. Verapamil did not affect the calcium rise induced by ACh in the absence of external sodium. The phorbol ester PMA (10 nM) caused a transient rise in calcium and inhibited the calcium rise caused by acetylcholine: it did not modify the effect of acetylcholine on inositol phosphates. The dependence of the stimulation of external calcium entry and inositol phosphate production on external sodium ions and protein kinase C is discussed.

The effects of thyrotropin-releasing hormone and potassium depolarization on phosphoinositide metabolism and cytoplasmic calcium in bovine pituitary cells.

Addition of thyrotropin-releasing hormone (TRH) (10 nM to 10 microM) to bovine anterior pituitary cells labelled with [3H]inositol decreased the radioactivity in inositol-containing lipids and increased it in inositol phosphates. TRH also increased the cytoplasmic calcium concentration biphasically. At TRH concentrations below 10 nM, the increase was sustained and sensitive to inhibitors of calcium influx through voltage-gated channels, whereas concentrations over 10 nM elicited in addition a rapid transient increase in calcium, which was relatively insensitive to such inhibition. Incubation of the cells in medium containing 25 mM KCl increased the cytoplasmic calcium concentration by stimulating influx through voltage-gated channels, and markedly enhanced the initial transient increase of calcium seen at TRH concentrations above 10 nM. It did not affect the generation of InsP3 and it also enhanced the calcium response to ionomycin. It is suggested that stimulation of calcium entry through voltage-gated channels can increase the amount of calcium available for mobilisation by TRH.

The effect of extracellular polyvalent cations on bovine anterior pituitary cells. Evidence for a Ca(2+)-sensing receptor coupled to release of intracellular calcium stores.

We have investigated the effects of extracellular cations ([ION]ex) on cytosolic free calcium levels ([Ca2+]i) in bovine anterior pituitary (bAP) cells, using single-cell microfluorimetry. Increasing the [Ca2+]ex from 1 mM to 20 mM caused [Ca2+]i to increase in 64 +/- 14% of bAP cells. The [Ca2+]ex-induced [Ca2+]i increase was observed when cells were maintained in the presence of the voltage-gated-calcium-channel antagonist nitrendipine, but not when cells were treated with thapsigargin. Addition of [La3+]ex (5-15 microM) decreased [Ca2+]i, whereas 30 microM-1 mM caused a [Ca2+]i rise in 60.9 +/- 8.8% of bAP cells. [La3+]ex-induced [Ca2+]i changes were abolished by treating bAP-cells with either thapsigargin or ionomycin, but not nitrendipine. [La3+]ex at 15 microM did not increase [Ca2+]i in any cells tested, but when cells were treated with thimerosal, [La3+]ex (15 microM) caused a [Ca2+]i increase in 62.5 +/- 12.2% of bAP cells. In the presence of 1 mM [Ca2+]ex, successive additions of La3+ caused successive [Ca2+]i rises, but in nominally [Ca2+]ex-free medium only the first addition of [La3+]ex caused a [Ca2+]i rise. Addition of thyroliberin (TRH) in the presence of 1 mM [Ca2+]ex, caused [Ca2+]i to increase in 70% of bAP cells; subsequent addition of [La3+]ex (1 mM) only caused [Ca2+]i increases in 75% of those cells which had already responded to TRH. However, all cells which responded to 1 mM [La3+]ex also responded subsequently to TRH. After treatment with TRH in medium that was nominally [Ca2+]ex free, addition of La3+ (0.5-1 mM) did not increase [Ca2+]i in any cells tested. The number of cells which showed [La3+]ex-induced [Ca2+]i increases decreased in culture: only 21.75 +/- 2.2% cells responded after 7-11 days. When cells were cultured for 7-11 days in the presence of tunicamycin, [La3+]ex failed to increase [Ca2+]i in any cells tested. [Mn2+]ex rapidly quenched the Fura-2 signal measured from all bAP cells, but at 10 mM it also triggered a [Ca2+]i rise in about 60% of bAP cells. The Mn(2+)-induced [Ca2+]i rise was specifically abolished in cells cultured in the presence of tunicamycin although quenching was still observed. From these data we suggest that bAP cells may express a polyvalent cation receptor coupled to the release of calcium from intracellular stores.

L-glutamate and acetylcholine mobilise Ca2+ from the same intracellular pool in cerebellar granule cells using transduction mechanisms with different Ca2+ sensitivities.

Ca2+ mobilisation induced by L-glutamate (Glu) and acetylcholine (ACh) has been studied in cultured cerebellar granule cells using digital fluorescence microscopy. The ability of Glu-receptor activation to mobilise Ca2+ was decreased when [Ca2+]o was lowered to 10 microM (from 1.8 mM). It was enhanced when [Ca2+]i was raised using 25 mM external K+ or by N-methyl-D-aspartate (NMDA), which selectively activates a distinct Glu-receptor subtype. The enhancement was dependent on entry of external Ca2+. In contrast, the ability of ACh receptor activation to mobilise Ca2+ was not affected by these conditions. Furthermore, pretreatment with pertussis toxin inhibited Ca2+ mobilisation in response to Glu-receptor activation without affecting mobilisation in response to ACh. However, activation of both receptors mobilised Ca2+ from a common, thapsigargin-sensitive pool. We conclude that there are differences in the Ca2+ mobilization pathways for the two receptor systems in cerebellar granule cells. The Ca(2+)-sensitivity of this Ca2+ mobilizing Glu receptor may have implications for its function in neuronal synaptogenesis and plasticity.

Ammonium ions mobilize calcium from an internal pool which is insensitive to TRH and ionomycin in bovine anterior pituitary cells.

The effects of NH4Cl on cytoplasmic free calcium concentration ([Ca2+]i) and pH (pHi) in single bovine anterior pituitary cells were determined using fluorescence imaging microscopy. Addition of NH4Cl (10-40 mM) in the presence of 1 mM extracellular calcium ([Ca2+]e) increased [Ca2+]i to a peak which then fell to a sustained plateau, returning to resting levels upon removal of NH4Cl. In medium containing 0.1 microM [Ca2+]e, or in 1 mM [Ca2+]e medium containing 0.1 microM nitrendipine, the plateau was absent leaving only a transient [Ca2+]i spike. NH4Cl also increased pHi and this, like the [Ca2+]i plateau, remained elevated during the continued presence of NH4Cl. In medium containing only 0.1 microM [Ca2+]e, to preclude refilling of internal stores by entry of external calcium, repeated exposures to NH4Cl induced repeated [Ca2+]i transients. In contrast, only the initial exposure to thyrotropin releasing hormone (TRH; 20-500 nM) caused a [Ca2+]i rise but, after an additional exposure to NH4CI, TRH responses re-emerged in some cells. Pre-treatment with the calcium ionophore ionomycin abolished the rise caused by TRH, but neither TRH nor ionomycin pretreatment affected the response to NH4Cl. Neither acetate removal nor methylamine increased [Ca2+]i in medium containing 0.1 microM [Ca2+]e, although in both cases pHi increased. We conclude that in bovine anterior pituitary cells NH4Cl raises [Ca2+]i by two independent pathways, increasing net calcium entry and mobilizing Ca2+ from a TRH-insensitive calcium store.

Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.

Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.

Use of a trapped fluorescent indicator to demonstrate effects of thyroliberin and dopamine on cytoplasmic calcium concentrations in bovine anterior pituitary cells.

The fluorescent calcium indicator 'quin2' was used to demonstrate changes in cytoplasmic calcium concentrations in bovine anterior pituitary cells. The basal calcium concentration was 0.21 +/- 0.02 microM (mean of 4 cell preparations). Thyroliberin (TRH) (10(-10) - 10(-6) M) rapidly and at the higher concentrations transiently increased the concentration. Dopamine (10(-10) - 10(-7) M) decreased the concentration transiently and more slowly. At 10(-5) M, dopamine prevented the increase in calcium concentration caused by 10(-9) M TRH, and partially inhibited the increase caused by higher concentrations of the peptide. The data support the hypothesis that calcium is the second messenger for TRH, and suggest that dopamine inhibits TRH-induced prolactin secretion by preventing the calcium concentration from exceeding the level necessary to increase secretion.

Inhibition by dopamine of 86Rb efflux and hormone secretion from bovine anterior pituitary cells perifused in the presence of acetylcholine or TRH.

The effects of dopamine (10(-5) M) on growth hormone and prolactin secretion and the fractional rate of 86Rb efflux were investigated using perifused bovine pituitary cells. Dopamine decreased the control efflux rate, prevented the rise caused by thyroliberin (10(-7) M) in the presence of 3-isobutyl-1-methylxanthine (10(-4) M) and greatly decreased the rise caused by acetylcholine (2.5 x 10(-5) M). Under these conditions it inhibited the prolactin secretion but it had no effect on growth hormone secretion induced by either treatment. Dopamine receptors in lactotrophs are therefore coupled to secretion and potassium permeability, but if somatotrophs contain dopamine receptors they are coupled only to K+ efflux.

Modification by pertussis toxin of the responses of bovine anterior pituitary cells to acetylcholine and dopamine: effects on hormone secretion and 86Rb efflux.

Acetylcholine is known to stimulate the secretion of growth hormone and prolactin and the efflux of 86Rb from bovine anterior pituitary cells: dopamine prevents the stimulation of 86Rb efflux and of prolactin but not growth hormone secretion. The sensitivity of these responses to pertussis toxin has been determined. Treatment of bovine anterior pituitary cells in primary culture with pertussis toxin (18 h, 100 ng/ml) did not modify the stimulation of prolactin secretion by acetylcholine, but prevented its inhibition by dopamine. In lactotrophs, dopamine but not acetylcholine receptors are therefore coupled to secretion through a pertussis toxin substrate. The stimulation of 86Rb efflux by acetylcholine was also unaffected by pertussis toxin and, again, its inhibition by dopamine was prevented. Treatment of the cells with pertussis toxin enhanced the secretion of growth hormone in response to acetylcholine. Nitrendepine (1 mumol/l) prevented the cholinergic stimulation of growth hormone but not prolactin secretion from these cells. Acetylcholine increased the cytoplasmic calcium concentration and this rise was enhanced by treatment of the cells with pertussis toxin. Nitrendepine partially inhibited the rise in calcium caused by acetylcholine, and prevented the enhancement of the rise following pertussis toxin treatment. Cholinergic stimulation of growth hormone therefore depends on calcium entry through nitrendepine-sensitive channels, whereas stimulation of prolactin secretion does not, and in somatotrophs a pertussis toxin substrate may limit calcium entry through these channels. These different sensitivities of somatotrophs and lactotrophs to pertussis toxin and nitrendepine may reflect differences in the properties of the predominant calcium currents in the two cell types.

Acetylcholine stimulates growth hormone secretion, phosphatidyl inositol labelling, 45Ca2+ efflux and cyclic GMP accumulation in bovine anterior pituitary glands.

Acetylcholine (25 micromol/l) in the presence of the choline esterase inhibitor physostigmine (67 micromol/l) increased the release of growth hormone and efflux of 45Ca2+ from perifused bovine pituitary slices; the time taken for the maximal response to occur was the same. In batch incubations, acetylcholine (1 micromol/l--1 mmol/l) increased pituitary cyclic GMP concentrations in the pituitary gland within 2 min, and increased incorporation of [3H]inositol and [32P]phosphate into pituitary phosphatidyl inositol within 15 min. Cyclic AMP concentrations were not significantly changed 2 or 5 min after acetylcholine addition. All the tissue responses were inhibited by prior exposure of the tissue to atropine (1 micromol/l) but not by tubocurarine (10 micromol/l--1mmol/l), indicating that the responses were mediated by receptors of the muscarinic type. The similarities between these responses and those to known hypothalamic hypophysiotrophic hormones are discussed.

Thyrotropin-releasing hormone-induced cytosolic calcium transients: characterisation of store refilling in bovine anterior pituitary cells.

The intracellular calcium ion concentration ([Ca2+]i) in individual bovine anterior pituitary cells was measured using fura-2 and ratiometric imaging. Addition of thyrotropin-releasing hormone (TRH) in the presence of external calcium ion ([Ca2+]e; 1 mM) caused a rapid transient increase in [Ca2+]i falling to a plateau which remained above pre-stimulation levels in the continued presence of TRH. Decreasing [Ca2+]e to 0.1 microM decreased [Ca2+]i. At 0.1 microM [Ca2+]e, the first TRH addition caused the rapid transient rise in [Ca2+]i but no plateau phase and a second addition of TRH did not cause a second transient rise. However, the second application of TRH in 0.1 microM [Ca2+]e caused a rise in [Ca2+]i if it was preceded by transient exposure of the cells to 2 mM [Ca2+]e. The presence of nitrendipine, 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBHQ), or TRH during the re-exposure to external calcium blocked this recovery of subsequent responses to TRH in the presence of only 0.1 microM [Ca2+]e. We conclude that refilling of the calcium stores depleted by TRH occurred only after the removal of agonist, used a tBHQ-sensitive uptake mechanism, and was mainly sustained by voltage-gated calcium entry into the cells.

Calcium-dependent protein phosphorylation in bovine anterior pituitary membranes and intact cells.

A calmodulin-activated protein kinase has been identified in bovine anterior pituitary membranes. This enzyme phosphorylated one endogenous substrate of subunit molecular weight 53,000 in the membranes. Phosphorylation of this protein was rapid, was half-maximal at 2.5 microM calcium in the presence of saturating concentrations of calmodulin (CaM), and was inhibited by trifluoperazine and thioridazine. A second protein was phosphorylated by an endogenous protein kinase in anterior pituitary membranes. Phosphorylation of this 42,000 Mr protein was reduced by calcium, was independent of exogenously added CaM, and was increased by trifluoperazine or thioridazine. The 42,000 Mr protein may be the alpha-subunit of pyruvate dehydrogenase. Calcium-dependent protein phosphorylation was also observed in intact cells; the largest increases were seen in proteins of Mr 42,000, 21,000 and 17,000.

Role of cyclic nucleotides in the inhibition of growth-hormone secretion by somatostatin.

The effects of somatostatin on GH secretion, cyclic AMP and cyclic GMp concentrations in dispersed bovine anterior pituitary cells were studied following activation of adenylate cyclase with cholera toxin and inhibition of phosphodiesterase with isobutylmethylxanthine (IBMX). Cholera toxin (10(-5)M) increased intracellular cyclic AMP concentration 10-fold and cyclic GMP concentration 3-fold relative to control, and stimulated the secretion of GH. IBMX (10(-4) M) als increased intracellular concentrations of both cyclic AMP and cyclic GMP and the secretion of GH and potentiated the actions of cholera toxin particularly in raising intracellular cyclic AMP concentrations which were elevated 40-fold in the presence of cholera toxin and IBMX. Somatostatin (5 X 10(-7) M) completely prevented GH secretion elicited by cholera toxin and/or IBMX. Somatostatin was without effect on control cyclic AMP and cyclic GMp concentrations and on the increases in both cyclic AMP and cyclic GMP caused by cholera toxin and by IBMX alone, or in combination. The data suggest that bovine GH secretion is increased when concentrations of either or both cyclic nucleotides are elevated within the cells, although incubation of cells with extracellular concentrations of cyclic AMP and cyclic GMP derivatives up to 2 x 10(-3) M caused only small changes in GH release. We suggest that somatostatin inhibits cholera toxin-induced bovine GH secretion by preventing activation of the secretory process by either cyclic AMP or cyclic GMP.

Effects of somatostatin and verapamil on growth hormone release and 45Ca fluxes.

Increasing the extracellular K+ concentration to 71 mM causes a phasic release of growth hormone and efflux of 45Ca from perifused bovine pituitary cells. Verapamil (20 micron) partially inhibits the initial phase of growth hormone release and 45Ca efflux and completely inhibits the second phase. Somatostatin (1 microgram/ml) partially inhibits both phases of growth hormone release but does not modify 5+-induced 45Ca efflux. Incubation of pituitary cells in 71 mM K+ increases 45Ca incorporation; verapamil (20 micron) completely prevents, and somatostatin (1 microgram/ml) partially inhibits, the K+-induced increase in 45Ca incorporation. The results suggest that 71 mM K+ increases both calcium entry into the cells and calcium redistribution within them, and that verapamil only inhibits the K+-induced calcium entry. Somatostatin may inhibit calcium entry into tissue stores.

Different sodium requirements for 86Rb efflux and for growth hormone and prolactin secretion from bovine anterior pituitary cells.

The sodium dependence of growth hormone and prolactin secretion and of 86Rb efflux from bovine anterior pituitary cells in response to acetylcholine and TRH was examined. Decreasing the external sodium concentration prevented the increases in the rates of 86Rb efflux and of growth hormone secretion caused by acetylcholine, or by TRH in the presence of IBMX. The growth hormone secretory response was less sensitive to sodium removal than was 86Rb efflux. However, even complete removal of extracellular sodium did not affect TRH-induced prolactin secretion. Ouabain and low extracellular potassium, which inhibit the sodium pump and increase intracellular sodium, prolonged the secretion of growth hormone in response to acetylcholine, but TRH-induced prolactin secretion was not affected. Inhibition of the sodium pump speeded the decline in the 86Rb efflux rate following stimulation by both acetylcholine and TRH. The results suggest that a sodium-dependent step is necessary for the efflux of 86Rb and for growth hormone secretion but not for prolactin secretion. The possible relationship between 86Rb efflux and hormone secretion from lactotrophs and somatotrophs is discussed.

Whole-cell patch-clamp recordings of an NMDA receptor-mediated synaptic current in rat hippocampal slices.

Whole-cell patch-clamp recordings and pharmacological techniques have been used to obtain low noise recordings of 2 excitatory postsynaptic synaptic currents (termed EPSCA and EPSCB) evoked by stimulation of the Schaffer collateral-commissural pathway in rat hippocampal slices. EPSCA was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and EPSCB was blocked by D-2-amino-5-phosphonovalerate (APV), indicating their mediation by non-N-methyl-D-aspartate (non-NMDA) and NMDA receptors, respectively. EPSCB has a slower time-course than EPSCA and its current-voltage relationship was highly non-linear with a region of negative slope conductance from -35 to -100 mV. These properties of EPSCA and EPSCB can explain their differing participation in synaptic transmission in this pathway.

Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells.

The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 microM), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K+. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.