Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Multiple Ca2+ channel-dependent components in growth hormone secretion from rat anterior pituitary somatotrophs.

The involvement of L-type Ca(2+) channels in both 'basal' and 'stimulated' growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non-L-type Ca(2+) channels is lacking. We investigated whether non-L-type Ca(2+) channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15 min with 2 mm K(+) ('basal' secretion) or 60 mm K(+) ('stimulated' secretion). The role of non-L-type Ca(2+) influx was investigated using specific channel blockers, including ω-agatoxin-IVA, ω-conotoxin GVIA or SNX-482, to block P/Q-, N- or R-type Ca(2+) channels, respectively. Our results demonstrate that P/Q-, N- and R-type Ca(2+) channels contributed 21.2 ± 1.9%, 20.2 ± 7.6% and 11.4 ± 1.8%, respectively, to 'basal' GH secretion and 18.3 ± 1.0%, 24.4 ± 5.4% and 14.2 ± 4.8%, respectively, to 'stimulated' GH secretion. After treatment with a 'cocktail' that comprised the previously described non-L-type blockers, non-L-type Ca(2+) channels contributed 50.9 ± 0.4% and 45.5 ± 2.0% to 'basal' and 'stimulated' GH secretion, respectively. Similarly, based on the effects of nifedipine (10 µM), L-type Ca(2+) channels contributed 34.2 ± 3.7% and 54.7 ± 4.1% to 'basal' and 'stimulated' GH secretion, respectively. Interestingly, the relative contributions of L-type/non-L-type Ca(2+) channels to 'stimulated' GH secretion were well correlated with the relative contributions of L-type/non-L-type Ca(2+) channels to voltage-gated Ca(2+) influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca(2+) channels is important for GH secretion. Lipid raft disruption (methyl-ß-cyclodextrin, 10 mm) abrogated the compartmentalisation of Ca(2+) channels and substantially reduced 'basal' and 'stimulated' GH secretion by 43.2 ± 3.4% and 58.4 ± 4.0%, respectively. In summary, we have demonstrated that multiple Ca(2+) channel-dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.

Somatostatin inhibits two types of voltage-activated calcium currents in rat growth-hormone secreting cells.

Somatostatin is known as the hypothalamic inhibitor of growth-hormone (GH) secretion from the pituitary gland. The present study examines the effects of somatostatin on voltage-gated calcium currents recorded from enriched populations of normal GH-secreting cells (somatotrophs). Two types of voltage-activated calcium currents were recorded from somatotrophs with the whole-cell mode of the patch-clamp technique. Somatostatin exerted a reversible blocking effect on these two types of calcium currents. These findings suggest that somatostatin can regulate intracellular calcium in somatotrophs by a direct control of calcium fluxes across the plasma membrane of these cells, thereby affecting the level of GH secretion.

Dopamine inhibits voltage-activated calcium channel currents in rat pars intermedia pituitary cells.

Several lines of evidence suggest that dopamine acts as a neurotransmitter that inhibits both hormone secretion and electrical activity in pituitary intermediate cells (melanotrophs). In this study we examined the effects of exogenously applied dopamine on voltage activated calcium currents recorded with the whole-cell mode of the patch-clamp technique from short-term primary cultures of melanotrophs. Two types of calcium currents were distinguished by their voltage dependence and kinetics of inactivation similar to the low voltage-activated currents (LVA; or T-type) and high voltage-activated currents (HVA; N&L-types) of calcium currents. Exogenously applied dopamine (2-20 microM) reversibly reduced both LVA and HVA types of calcium currents. Evidence for these results came from experiments in which LVA and HVA calcium currents were separated by stepping to different membrane potentials from a fixed holding potential (Vh) or by changing Vh. These results suggest that dopamine can regulate the entry of calcium into melanotrophs by acting on at least two different populations of calcium channels thereby affecting hormone secretion and electrical activity.

Noradrenaline augments tetanic potentiation of transmitter release by a calcium dependent process.

Noradrenaline (25 microM-50 microM) causes an increase in tetanic potentiation and in the augmentation phase of posttetanic potentiation of miniature and plate potential frequency. These effects were observed at both the frog and the rat neuromuscular junctions. The action of noradrenaline on quantal transmitter release depends on the presence of calcium ions in the extracellular medium.

Multiple pathways for high voltage-activated ca(2+) influx in anterior pituitary lactotrophs and somatotrophs.

The present study demonstrates that a significant proportion of high voltage-activated (HVA) Ca(2+) influx in native rat anterior pituitary cells is carried through non-L-type Ca(2+) channels. Using whole-cell patch-clamp recordings and specific Ca(2+) channel toxin blockers, we show that approximately 35% of the HVA Ca(2+) influx in somatotrophs and lactotrophs is carried through Ca(v) 2.1, Ca(v) 2.2 and Ca(v) 2.3 channels, and that somatotrophs and lactotrophs share similar proportions of these non-L-type Ca(2+) channels. Furthermore, experiments on mixed populations of native anterior pituitary cells revealed that the fraction of HVA Ca(2+) influx carried through these non-L-type Ca(2+) channels might even be higher (approximately 46%), suggesting that non-L-type channels exist in the majority of native anterior pituitary cells. Using western blotting, immunoblots for α(1C) , α(1D) , α(1A) , α(1B) and α(1E) Ca(2+) channel subunits were identified in native rat anterior pituitary cells. Additionally, using reverse transcriptase-polymerase chain reaction, cDNA transcripts for α(1C) , α(1D) , α(1A) and α(1B) Ca(2+) channel subunits were identified. Transcripts for α(1E) were nonspecific and transcripts for α(1S) were not detected at all (control). Taken together, these results clearly demonstrate the existence of multiple HVA Ca(2+) channels in the membrane of rat native anterior pituitary cells. Whether these channels are segregated among different membrane compartments was investigated further in flotation assays, demonstrating that Ca(v) 2.1, Ca(v) 1.2 and caveolin-1 were mostly localised in light fractions of Nycodenz gradients (i.e. in lipid raft domains). Ca(v) 1.3 channels were distributed among both light and heavy fractions of the gradients (i.e. among raft and nonraft domains), whereas Ca(v) 2.2 and Ca(v) 2.3 channels were distributed mostly among nonraft domains. In summary, in the present study, we demonstrate multiple pathways for HVA Ca(2+) influx through L-type and non-L-type Ca(2+) channels in the membrane of native anterior pituitary cells. The compartmentalisation of these channels among raft and nonraft membrane domains might be essential for their proper regulation by separate receptors and signalling pathways.

Growth hormone releasing factor evokes rhythmic hyperpolarizing currents in rat anterior pituitary cells.

1. The effect of human pancreatic growth hormone releasing factor (hpGHRF) on the electrical activity of dissociated rat anterior pituitary cells in culture was studied, using both the cell-attached and whole-cell modes of the patch-clamp recording technique. 2. To avoid possible wash-out of the responses, extracellular records were made from cell-attached patches. Application of hpGHRF to the cells produced rhythmic inward currents through the patches, attributable to rhythmic hyperpolarizations of the cell membrane outside the patch. The amplitude of the current oscillations was 1-8 pA and the frequency 0.05-0.4 Hz. 3. Flooding the cells with K+ ions from a small pipette containing 50 mM or 100 mM-K+ resulted in a reversible attenuation or block of the rhythmic inward currents evoked by hpGHRF, indicating that changes in K+ conductance were involved in the responses. 4. Flooding the cells with a solution containing 10 mM-EGTA blocked these rhythmic inward currents reversibly, suggesting the involvement of Ca2+ in the responses. In addition, responses were blocked by adding Co2+ (5-10 mM) to the bathing medium. The presence of tetrodotoxin (3 microM) had no effect, ruling out the participation of voltage-gated Na+ channels. 5. With whole-cell recording, the resting potential (-41.46 +/- 7.78 mV) and input resistance (5.34 +/- 3.73 G omega) of anterior pituitary cells in culture were found to be similar to those previously reported for pituitary cells and chromaffin cells with the same recording method. 6. In whole-cell experiments, application of hpGHRF (shortly prior to intracellular penetration) evoked rhythmic outward currents, associated with conductance increases, when the cells were clamped at their resting potential. The persistence of these currents in the 'voltage-clamped' cell indicated that the rhythmicity was not related to voltage-dependent phenomena. The currents disappeared within 4 min after breaking into the cell, presumably because of 'washout' of cell constituents into the pipette. 7. The reversal potential (-60 mV) of the hpGHRF-induced currents was negative to the resting potential of the cells (-41 mV), further indicating that hpGHRF would evoke rhythmic hyperpolarizations in unclamped cells, possibly due to periodic increases in K+ conductance. 8. The possible relation of these rhythmic currents to hpGHRF-induced secretion of growth hormone is discussed.

Ionic basis of tetanic and post-tetanic potentiation at a mammalian neuromuscular junction.

1. The ionic basis of tetanic and post-tetanic potentiation (TP and PTP) was studied at the rat soleus neuromuscular junction (NMJ), using the miniature endplate potential (MEPP) frequency as an index for transmitter release. Conventional intracellular recording and computer-assisted data analysis were employed. 2. The experimental results in this study indicate that contrary to previous suggestions, there is a substantial similarity in the ionic basis of TP and PTP at the mammalian and amphibian motor nerve terminals which can be subdivided into [Ca2+]o-dependent and [Ca2+]o-independent parts. 3. Tetanic and post-tetanic increase in MEPP frequency at the rat soleus NMJ is similar to that at the frog NMJ in the following aspects: (i) Tetanic potentiation is substantially larger in calcium-containing solutions than in calcium-deficient solutions. About 90% of tetanic potentiation is contributed by extracellular calcium. (ii) Increase in [Mg2+]o reduces tetanic potentiation in calcium-containing solutions and enhances TP in calcium-defient solutions. Elevated [Mg2+]o prolongs the post-tetanic potentiation both in calcium-containing and in calcium-deficient solutions. (iii) A post-tetanic jump in MEPP frequency was observed in 44% of the experiments performed in calcium-deficient solutions. (iv) The augmentation phase of post-tetanic potentiation, evident in calcium-containing solutions, is completely abolished by removal of [Ca2+]o. (v) Tetanic and post-tetanic potentiations are enhanced by increasing the rate and duration of tetanic stimulation in calcium-containing solutions. 4. The [Ca2+]o-independent part of tetanic potentiation is presumably due to entry of sodium ions and their accumulation in the nerve terminal, since it is increased by measures known to inhibit the sodium pump: reduction in [K+]o and partial substitution of sodium by lithium. 5. Sodium ions contribute substantially to the [Ca2+]o-independent part of posttetanic potentiation, since its duration is markedly prolonged by ouabain, reduction in [K+]o and partial substitution of sodium by lithium. 6. Tetanic potentiation is manifested earlier in calcium-containing media than in calcium-deficient media. This difference may indicate that sodium entry into the terminal during tetanic stimulation is at locations remote from the releasing sites. Alternatively, this time difference may be due to the delay between intracellular sodium accumulation and the increase in transmitter release.

Activity-dependent ultra-slow inactivation of calcium currents in rat anterior pituitary cells.

1. The inactivation of high-voltage-activated (HVA) calcium currents during long depolarizations in holding potential (Vh) was studied with the use of whole cell patch-clamp recording from rat anterior pituitary cells. 2. An ultra-slow inactivation in the amplitude of HVA calcium currents, with an average slow time constant of 149.3 s for peak currents and 159.1 s for sustained currents (n = 9), was unveiled during 5-min step depolarizations in Vh. 3. The ultra-slow inactivation of HVA calcium currents was found to be generated by at least two processes: a voltage-dependent inactivation that increases with increasing depolarization in Vh and an activity-dependent inactivation that is initiated, but not increased, with increasing depolarization in Vh. The relative contribution of the activity-dependent component to the ultra-slow inactivation was 80% when Vh was stepped from -80 to -60 mV and only 40% when Vh was stepped from -80 to -40 mV. 4. The activity-dependent inactivation of the HVA currents was not altered significantly in experiments in which barium replaced calcium as charge carrier and 1,2-bis (1-aminophenoxy) ethane N,N,N' N'-tetraacetic acid (BAPTA) was used as an intracellular calcium buffer instead of the less potent ethylenglycol-bis-(beta-aminoethylether) N,N,N' N'-tetraacetic acid (EGTA). In addition, activity-dependent inactivation was observed with sodium as the charge carrier through the calcium channels. 5. The activity-dependent inactivation depends on divalent cation influx. The activity-dependent inactivation was abolished when the test potentials, during the depolarization in Vh, were increased from 0 to +70 mV (close to the reversal potential for calcium currents under our experimental conditions). This reduction in driving force for calcium currents eliminated divalent cation influx and abolished the activity-dependent inactivation. 6. The activity-dependent inactivation lacks several characteristic features for calcium-dependent inactivation, such as dependence on charge carrier (see above), dependence on the size of the calcium current, and increase in decay rate of the calcium current during the test pulse. These latter notions were also supported by our paired pulse experiments, in which the calcium current elicited by a constant test pulse was virtually unaffected (7%) by conditioning pulses that produced maximal calcium currents. We therefore conclude that the dependence of activity-dependent inactivation on divalent cation influx cannot be attributed to the known form of calcium-dependent inactivation. 7. In conclusion, this study shows that calcium influx through HVA channels in anterior pituitary cells can be regulated by subthreshold changes in membrane potential and that the extent of this regulation depends on low-frequency activation of HVA calcium channels during the depolarization in membrane potential. Thus the pituitary cell may regulate hormone secretion by changes in membrane potential and in a use-dependent manner via regulation of calcium influx.

Mechanosensitivity of voltage-gated calcium currents in rat anterior pituitary cells.

Sensitivity of voltage-activated calcium currents to flow-induced mechanical stress was examined in enriched populations of rat anterior pituitary somatotrophs. Voltage-activated calcium currents were recorded with the whole-cell configuration of the patch-clamp technique. Pituitary cells were exposed to flow (from pipettes) which was produced by a hydrostatic pressure of about 3 cmH2O. In 92% of the cells studied (n = 87 cells) flow reduced the amplitude of both low voltage-activated (LVA) and high voltage-activated (HVA) calcium currents. These effects of flow on calcium currents did not result from changes in either seal resistance or leak conductance of the cell and were dependent on the magnitude of flow. The effect of flow is selective. We found that LVA calcium currents were substantially more sensitive to flow than HVA calcium currents. Under constant flow conditions, LVA calcium currents were reduced by 57.6 +/- 29.6% (S.D.), whereas HVA currents (recorded from the same cells) were reduced by only 17.8 +/- 15.9% (S.D.). The effects of flow on calcium currents were associated with effects on their related calcium tail currents. Slowly deactivating calcium tail currents were reduced by 75.3 +/- 25.6% (S.D.), whereas rapidly deactivating calcium tail currents were reduced by 29.1 +/- 14.4% (S.D.). The effect of flow on calcium currents was not associated with any significant shift in the activation curves of the calcium currents (voltage range -60 to +30 mV), suggesting that the effect of flow is not voltage dependent. The effect of flow is not dependent on activation of calcium currents during the exposure to flow. Calcium currents which were evoked immediately after cessation of the exposure to flow were reduced in amplitude and recovered to control values. Possible mechanisms underlying the flow effect and possible physiological relevance of the effect on pituitary cells are discussed.

Hyperosmotic regulation of voltage-gated calcium currents in rat anterior pituitary cells.

1. The sensitivity of voltage-gated calcium currents to hyperosmotic media containing mannitol or sucrose (373-723 mOsm) and to the dihydropyridine (DHP) calcium channel agonist Bay K 8644 was examined in enriched populations of rat anterior pituitary somatotrophs by using the whole cell mode of the patch-clamp technique. 2. Hyperosmotic media reduced the amplitude of voltage-gated calcium currents. With a 61.9% increase in extracellular medium osmolarity (523 mOsm), low voltage-activated (LVA) calcium currents were reduced to 67.9 +/- 17.8% of control size and high voltage-activated (HVA) calcium currents were reduced to 57.0 +/- 5.7% (mean +/- SD) of control size. The hyperosmotic suppression of HVA calcium currents was usually accompanied with a negative shift of 6.0 +/- 2.9 mV (mean +/- SD) in the activation curve of HVA currents. 3. The DHP calcium-channel agonist Bay K 8644 (10 microM), which stimulates hormone secretion from somatotrophs, increased the amplitude of HVA calcium currents to 212.6 +/- 67.2% of their control size, prolonged their tail currents, and negatively shifted the activation curve of HVA calcium currents by 6.2 +/- 2.8 mV. 4. Hyperosmotic media reduced the amplitude of DHP-sensitive HVA calcium currents and their associated prolonged tail currents, thus providing direct evidence for hyperosmotic suppression of DHP-sensitive currents. 5. Hence, exposure of pituitary cells to hyperosmotic media reduced voltage-sensitive calcium influx through LVA and DHP-sensitive HVA calcium channels. The inhibition of calcium influx through DHP-sensitive channels, which are implicated in regulation of hormone secretion in these cells, suggests that inhibitory hyperosmotic effects on hormone secretion from pituitary cells may stem from inhibition of calcium influx, before the exocytotic process. These results may also be relevant to effects of hypertonicity on neurosecretion in the nervous system.

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch-clamp capacitance, FM1-43 fluorescence imaging and amperometry. Stimulation of exocytosis through voltage-dependent Ca2+ channels by depolarizations (1-5 s) increased the capacitance by 4.3 +/- 0.9 % and the fluorescence by 6.6 +/- 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane-bound fluorescent dye FM1-43 in a quantitatively similar manner. Intracellular dialysis (0.5-4.5 min) with elevated Ca2+ (1.5-100 microM) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine-loaded cells (10 cells) or as an increase in FM1-43 fluorescence (56 +/- 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 +/- 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. Sustained endocytosis was not blocked when the intracellular GTP (300 microM) was replaced with GTP[gamma]S. Replacing intracellular Ca2+ (100 microM) with Ba2+ (300 microM) or Sr2+ (200 microM), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole-cell recordings.

Regulation of acetylcholine liberation from presynaptic nerve terminals.

Acetylcholine is liberated from motor nerve terminals either as a molecular leakage or as quantal packages; the latter form of release is responsible for signaling across the neuromuscular synapse. Three main factors determine the number of quanta liberated by the nerve impulse: the degree of presynaptic depolarisation, the frequency of activation of the nerve terminal, and calcium ion concentration in the extracellular medium. These factors seem to act yb changing the free calcium ion concentration [Ca]in in the presynaptic nerve terminal. Thus, processes that change [Ca]in will determine efficiency of synaptic transmission. These processes include fluxes of calcium ions across the presynaptic membrane and reversible translocation by intracellular organelles such as mitochondria, vesicles and soluble molecules. The level of intracellular [Ca] can be changed by ion-containing liposomes. One of the main physiological determinants of the level of transmitter release is potentiation, where the increase in transmitter release is caused by transmembranal processes and intracellular translocation.