On the involvement of cyclic AMP and extracellular Ca2+ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture

Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-l-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH 3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM–1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive.The Ca²⁺ channel blockers Co²⁺ (5 mM) and verapamil (100 M) and the Ca²⁺ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 M) on hormone release. Co²⁺ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co²⁺ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co²⁺, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co²⁺, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co²⁺ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co²⁺, was increased by TRH.The calmodulin antagonist trifluoperazine (TFP) at 30 M inhibited basal hormone release and hormone release stimulated by TRH (1 M), db-cAMP (5 mM), and MIX (1 mM). The Ca²⁺ ionophore A23187 (5 M) had a stimulatory effect on basal hormone release which was abolished by 30 M TFP.     

Intracellular free sodium concentrations in GH4C1 cells

In the present investigation, intracellular sodium ([Na⁺]_i) levels were determined in GH₄C₁ cells using the fluorescent probe SBFI. Fluorescence was determined by excitation at 340 nm and 385 nm, and emission was measured at 500 nm. Intracellular free sodium ([Na⁺]_i) was determined by comparing the ratio 340/385 to a calibration curve. The ratio was linear between 10 and 60 mM Na⁺. Resting [Na⁺]_i in GH₄C₁ cells was 26 ± 6.2 mM (mean ± SD). In cells incubated in Na⁺-buffer [Na⁺]_i decreased to 3 ± 3.6 mM. If Na⁺/K⁺ ATPase was inhibited by incubating the cells with 1 mM ouabain, [Na⁺]_i increased to 47 ± 12.8 mM in 15 min. Stimulating the cells with TRH, phorbol myristyl acetete, or thapsigargin had no effect on [Na⁺]_i. Incubating the cells in Ca²⁺-buffer rapidly increased [Na⁺]_i. The increase was not inhibited by tetrodotoxin. Addition of extracellular Ca²⁺, nimodipine, or Ni²⁺ to these cells immediately decreased [Na⁺]_i, whereas Bay K 8644 enhanced the influx of Na⁺. In cells where [Na⁺]_i was increased the TRH-induced increase in intracellular free calcium ([Ca²⁺]_i) was decreased compared with control cells. Our results suggest that Na⁺ enters the cells via Ca²⁺ channels, and [Na⁺]_i may attenuate TRH-induced changes in [Ca²⁺]_i in GH₄C₁ cells. © 1993 Wiley-Liss, Inc.     

On the role of extracellular Ca2+ for prolactin release and adenosine 3′:5′-monophosphate formation induced by thyroliberin in cultured rat pituitary cells

In cultured rat pituitary tumour cells (GH₃ cells) the absence of extracellular Ca⁺⁺ or addition of NaEGTA reduced spontaneous prolactin (PRL) release and abolished the stimulatory effect of thyroliberin (TRH). Readdition of CaCl₂, but not of equimolar concentrations of MgCl₂ increased spontaneous hormone release, and restored the effect of TRH. The calcium ionophore, A-23187, induced PRL release during normal calcium conditions, but not when an excess NaEGTA was present. TRH increased cyclic AMP accumulation in the presence and the absence of extracellular calcium. The effect of TRH on PRL release and cyclic AMP formation occured concomitantly with an increased efflux of ⁴⁵Ca²⁺. Intracellular electrophysiological recordings from the same single cells before and after TRH activation showed increased frequency and duration of the Ca²⁺ dependent action potentials. We conclude that TRH elevates the Ca²⁺ influx which depends on the depolarizing action current, and this effect is probably linked to formation of cyclic AMP and PRL release.     

Effects of caffeine on the influx of extracellular calcium in GH4C1 pituitary cells

Caffeine increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular Ca²⁺ stores. Caffeine also can change [Ca²⁺]_i by affecting Ca²⁺ influx through voltage-operated Ca²⁺ channels (VOCCs). In the present study, we investigated the effects of caffeine on Ca²⁺ entry in GH₄C₁ pituitary cells. Pretreatment of the cells with caffeine attenuated the high K⁺-evoked influx of ⁴⁵Ca²⁺ in a dose-dependent manner. This inhibition was not secondary to the caffeine-evoked elevation of [Ca²⁺]_i because caffeine was able to inhibit VOCCs also in the presence of the intracellular Ca²⁺ chelator BAPTA. However, the inhibitory effect of caffeine on ⁴⁵Ca²⁺ entry appeared to be dependent on the degree of depolarization of the plasma membrane. Only in cells depolarized with relatively high concentrations of K⁺ (20, 35, and 50 mM) was the caffeine-induced inhibition observed. A similar inhibitory effect of caffeine on the high K⁺-evoked calcium and barium entry was observed in experiments using Fura 2. Neither IBMX, forskolin nor dibutyryl cAMP reduced the enhanced [Ca²⁺]_i induced by 50 mM K⁺, suggesting that the effect of caffeine was not due to increased intracellular cAMP. Furthermore, high doses of caffeine inhibited the plateau level of the TRH-induced increase in [Ca²⁺]_i, which is caused partly by influx of Ca²⁺ through VOCCs. The inhibitory effect of caffeine was, in part, due to an hyperpolarization of the plasma membrane observed at high doses of caffeine. On the other hand, low doses of caffeine enhanced depolarization-evoked Ba²⁺ entry as well as the TRH-evoked plateau level of [Ca²⁺]_i. We conclude that caffeine has a dual effect on Ca²⁺ entry through activated VOCCs in GH₄C₁ cells: at low concentrations caffeine enhances Ca²⁺ entry, whereas high concentrations of caffeine block Ca²⁺ entry. J. Cell. Physiol. 171:52–60, 1997. © 1997 Wiley-Liss, Inc.     

Interaction of calcium and cyclic nucleotides in thyroliberin-stimulated prolactin release

The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH₃ and GH₄ rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH₃ and GH₄ cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH₃ or GH₄ cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH₃ or GH₄ cells.     

Glucose-induced mobilisation of intracellular Ca2+ in depolarised pancreatic islets

Perifused rat pancreatic islets, prelabelled with ⁴⁵Ca, were exposed for 90 min to a medium containing 30 mM K⁺, 0.25 mM diazoxide and 0.5 mM EGTA, but deprived of CaCl₂. Either verapamil (0.05 mM) or Cd²⁺ (0.05 mM) were also present in the perifusate. Under these conditions a rise in D-glucose concentrations from either 2.8 to 16.7 mM or zero to 8.3 mM increased both ⁴⁵Ca outflow and insulin release, after an initial and transient decrease in effluent radioactivity. These findings suggest that, in islets depolarised by exposure to a high extracellular concentration of K⁺, D-glucose provokes an intracellular redistribution of Ca²⁺ ions and subsequent stimulation of insulin release. The functional response to D-glucose is apparently not attributable to either the closing of ATP-sensitive K⁺ channels, which were actually activated by diazoxide, or stimulation of Ca²⁺ influx, which was prevented by the absence of extracellular Ca²⁺. The present experimental design thus reveals a novel component of the glucose-induced remodelling of Ca²⁺ fluxes in islet cells. Such an effect might also be operative under physiological conditions, when the hexose leads to depolarisation of the islet B-cells.     

Effects of colchicine and 2-Br-alpha-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone by functional pituitary tumor cells in culture

The effects of colchicine and 2-Br-α-ergocryptine-methane-sulfonate (CB 154) on the release of prolactin and growth hormone have been studied in a clonal strain of rat pituitary tumor cells (GH₃) in monolayer culture. These cultures produce both prolactin and growth hormone and release both proteins spontaneously into the medium without storing them in large amounts. Immunological methods were used to measure both intracellular and extracellular concentrations of the hormones. Colchicine (5 × 10^?6 M for 3 hours) caused a 2- to 3-fold increase in intracellular concentrations of prolactin and growth hormone but, under basal conditions, had little or no measurable effect on the amounts of hormone accumulated in the medium during the course of the standard three hour treatment period. This latter finding evidently is due to a lag in the onset of drug action. Colchicine had little or no effect on accumulation of extracellular prolactin during the first two hours of treatment whereas such accumulation was depressed by over 60% during the third hour of treatment. Previous studies have shown that treatment of GH₃ cells with thyrotropin releasing hormone (TRH) and hydrocortisone (HC) increases both intra and extracellular levels of prolactin and growth hormone, respectively. In cultures treated with TRH (5 × 10^?8 M), colchicine (5 × 10^?6 M for 3 hours) increased intracellular prolactin by about 70% and decreased extracellular hormone by 10%. In cultures treated with HC (3 × 1O^?6 M), colchicine increased intracellular growth hormone by more than 100% and decreased medium concentrations of the hormone by 15%. Colchicine did not significantly alter total hormone (intracellular + extracellular) accumulation, cellular uptake of ³H-amino acids, or total cell protein synthesis. The synthetic ergot alkaloid, CB 154, (3.3 × 10^?6 M for 3 hours) caused an 80% increase in intracellular, and a nearly 50% decrease in extracellular, prolactin without affecting the accumulation of growth hormone, the uptake of ³H-labeled amino acids, or overall protein synthesis in the cultures. Elevation of medium potassium concentration from a basal value of 5.3 mM to 3–5 × 10^?2 M (by addition of KCl) decreased intracellular levels of prolactin by 85% and growth hormone by 55%. These effects of high potassium were blocked by colchicine and by CB 154. We conclude that colchicine, after a lag period of two hours, acts to inhibit the release of prolactin and growth hormone from GH₃ cells. By the end of three hours of treatment, this inhibition is over 60% complete in the case of prolactin. The qualitatively different effects of colchicine and CB 154 on prolactin and growth hormone release suggest that these two secretory blocking agents probably act on GH₃ cells by different mechanisms.     

THE ROLE OF INTRANEURONAL 5-HT AND OF TRYPTOPHAN HYDROXYLASE ACTIVATION IN THE CONTROL OF 5-HT SYNTHESIS IN RAT BRAIN SLICES INCUBATED IN K+-ENRICHED MEDIUM

Abstract— The incubation of brain stem slices from adult rats in a K⁺-enriched medium containing a 5-HT uptake inhibitor (fluoxetine) significantly increased their capacity to synthesize 5-HT from tryptophan. The K+-induced stimulation of 5-HT synthesis was at least partly dependent on the depletion of the indoleamine in tissues since: (1) a good correlation was found between the respective changes in 5-HT release and synthesis evoked by high K⁺ concentrations in the presence of various 5-HT uptake inhibitors; (2) the modifications in endogenous 5-HT levels produced by in vim treatments with drugs (reserpine, pargyline) or by incubating slices with 5-HT altered the stimulating effect of high K⁺ concentrations and fluoxetine on 5-HT synthesis; (3) the replacement of Ca²⁺ by Co²⁺ (4 mM) or EGTA (0.1 mM) in the incubating medium completely prevented the increased 5-HT release and synthesis evoked by high K⁺ concentrations and fluoxetine. The extraction of tryptophan hydroxylase from incubated tissues revealed that the increased 5-HT synthesis occurring in K⁺-enriched medium was associated with an activation of this enzyme. Kinetic analyses indicated that this activation resulted from an increase in the V_max of tryptophan hydroxylase, its apparent affinities for both tryptophan and 6-MPH₄ being not significantly affected. In contrast to the tryptophan hydroxylase from tissues incubated in normal physiological medium, the activated enzyme from tissues depolarized by K⁺ was hardly stimulated by Ca²⁺-mediated phosphorylating conditions. This led to the proposition of a hypothetical model by which the Ca²⁺ influx produced by the neuronal depolarization would trigger the activity of a Ca²⁺-dependent protein kinase capable of activating tryptophan hydroxylase. Although this sequence is still largely speculative it must be emphasized that, as expected from such a model, the regional differences in the K⁺-evoked activation of tryptophan hydroxylase in slices (cerebral cortex > brain stem > spinal cord) were parallel to those of the Ca²⁺-dependent protein phosphorylation (r= 0.92) and those of the activating effect of phosphorylating conditions on soluble tryptophan hydroxylase (r= 0.96).     

Basal and potassium stimulated, calcium dependent, endorphins release from pituitary cells.

Mouse pituitary tumor cells grown in tissue culture release endorphins spontaneously to the culture medium. Depolarization of these cells by incubation with high K⁺ concentration (56 mM) increased 2–3 folds the release of endorphins. The K⁺ evoked release was Ca⁺⁺ dependent by that: $\text{a}$, removal of Ca⁺⁺ ions inhibited 90% of K⁺ stimulated release. $\text{b}$, ethyleneglycol-bis (β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) inhibited release of endorphins in the presence of high K⁺ and Ca⁺⁺. It is suggested that dual regulatory system inhibit and/or stimulate $\text{in-vivo}$ release of endorphins from the pituitary glands.     

Defective thyroliberin-induced prolactin synthesis and release in a hybrid GH strain

Summary The hybrid GH cell strain, 928-9b, isolated from PRL+ (prolactin [PRL] producing) GH₄Cl and PRL (PRL non-producing) FIBGH12CI cells, has specific TRH (thyroliberin) receptors, yet does not respond to this peptide hormone. Unlike the parent strain, GH₄Cl, TRH does not stimulate synthesis or release of PRL in the hybrid strain. In contrast, treatment of 928-9b cells with another peptide, EGF (epidermal growth factor), stimulates both release and synthesis of PRL. The number of EGF receptors in the hybrid strain (2.5 × 10³/cell) and the affinity of these receptors for ligand (2.2 nM) are comparable to that of the parent strain, GH₄C₁. The EGF dose response curve is also essentially the same for parent and hybrid cells for the enhancement of PRL production. A 3-8-fold enhancement of PRL production is observed and 1/2 maximal enhancement occurs at approximately 5 × 10¹¹ M EGF for both strains. TRH does not have any potentiating effect on EGF-induced stimulation of PRL release or PRL synthesis in the hybrid strain. Although EGF and TRH have similar biological effects in responsive GH cells, binding of one hormone to its receptors does not modulate the binding of the heterologous hormone. These findings demonstrate that more than one effect of TRH is defective in 928-9b cells even though EGF responses are intact. This suggests that 1) TRH-stimulated PRL release and TRH-stimulated PRL production have a common intermediate step, and 2) TRH and EGF have a different mechanism of action in GH cells.     

Growth hormone-releasing factor reduces voltage-gated Ca2+ channel current in Rat GH3 cells

Summary The action of GRF on GH₃ cell membrane was examined by patch electrode techniques. Under current clamp with patch elecrtrode, spontaneous action potentials were partially to totally eliminated by application of GRF. In the case of partial elimination, the duration of remaining spontaneous action potentials was prolonged and the amplitude of afterhyperpolarization was decreased. The evoked actiion potential in the cells which did not show spontaneous action potentials was also eliminated by GRF. In order to examine what channels were affected by GRF, voltage-clamp analysis was performed. It was revealed that voltage-gated Ca²⁺ channel current and Ca²⁺-induced K⁺ channels current were decreased by GRF, while voltage-gated Na⁺ channel and delayed K⁺ channel current was considered to be a consequence of he decrease of voltage-gated Ca²⁺ channels current. Therefore it is likely that the effect of GRF on GH₃ cells was due to the block of voltage-gated Ca²⁺ channels. The elimination of action potential under current clamp corresponded to the block of voltage-gated Ca²⁺ channels and the prolongation of action potential could be explained by the decrease of Ca²⁺-induced K⁺ channel current. The amplitude decrease of afterhyperpolarization could also be explained by the reduction of Ca²⁺-induced K⁺ channel current. Thus the results under current clamp well coincide with the results under voltage clamp. Hormone secretion from GH₃ cells was not stimulated by GRF. However, the finding that GRF solely blocked voltage-gated Ca²⁺ channel suggested the specific action of GRF on GH₃ cell membranes.     

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K⁺ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K⁺ concentration produced a graded release of ATP from synaptosomes. Rb⁺ also released ATP but Na⁺, Li⁺ and choline did not. The response to elevated K⁺ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na⁺-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K⁺. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na⁺-channels was involved. Neither veratridine nor elevated K⁺ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K⁺ was diminished in a medium lacking CaCl₊ or when EGTA was added to chelate Ca²⁺. In contrast, release by veratridine appeared to be augmented in Ca²⁺-free media or in the presence of EGTA. The K⁺-induced release of ATP, which is Ca²⁺ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca²⁺ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.     

The calcium requirement for stimulation of rubidium efflux from isolated rat parotid acinar cells by carbachol.

Stimulation of ⁸⁶Rb⁺ efflux from isolated parotid acinar cells by carbchol was biphasic. The phases of stimulated ⁸⁶Rb⁺ efflux were separated on the basis of their relative requirements for extracellular Ca²⁺. If the isolated cells were incubated in Ca²⁺ free buffer containing 1.0 mM ethylene glycol bis (β-aminoethyl ether) N, N¹ - tetra acetic acid (EGTA) for 30 min. before adding carbachol an initial phase of ⁸⁶Rb⁺ efflux was observed. A second phase of ⁸⁶Rb⁺ efflux was obtained upon addition of 2.0 mM Ca²⁺. However when cells were incubated for 60 min. in Ca²⁺ free buffer containing 1.0 mM EGTA the initial phase of release caused by carbachol was inhibited by 95 percent. If the EGTA was titrated with Ca²⁺ to give 1.0 mM Ca²⁺, following the 60 min. depletion regimen, the second phase was observed. Although 60 min. of Ca²⁺-depletion in EGTA buffer was required for complete inhibition of the effect of carbachol on the initial phase of ⁸⁶Rb⁺ efflux, the response was fully restored within 4 min. after the readdition of Ca²⁺.     

Stimulation-evoked release of purines from the rabbit retina

The evoked release of purines from rabbit retinae preloaded with [³H]adenosine was studied in vitro. Potassium (8.6–43.6 mM) and ouabain (1 or 10 μM) increased the release of radioactivity in a concentration-dependent manner. The K⁺-evoked release was significantly reduced when the superfusion was carried out at 2–4°C. The effect of K⁺ (8.6, 13.6 and 23.6 mM) and of ouabain (1 μM) were completely abolished when the retinae were superfused with a Ca²⁺-free medium containing 0.1 mM EGTA. Calcium removal only partially reduced the effect of higher K⁺ and ouabain concentrations (43.6 mM and 10 μM, respectively). Further, the effect of K⁺ was found to be independent of extracellular Ca²⁺ when retinae were pretreated with ouabain for 30 min. Stimulation of the retina with light flashes induced a small, persistent increase in the release of radioactivity observable for several minutes after the end of stimulation.The superfusate contained mainly hypoxanthine and inosine. There were no significant changes in the relative proportions of the different purine compounds released before or in response to either K⁺ (23.6 mM) or ouabain (10 μM) stimulation. Potassium stimulation significantly increased the release of adenosine, inosine and hypoxanthine. Addition of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), significantly increased the relative proportions of released endogenous adenosine and inosine.The results indicate that K⁺ stimulation induces the release of purines from the rabbit retina by a Ca²⁺- and energy-dependent process. Light flashes also induce a purine release. The results suggest an active role for adenosine in retinal neurotransmission.     

Changes in free-calcium levels and pH in synaptosomes during transmitter release

The free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in rat brain synaptosomes estimated by the indicator quin 2 is 104±8 nM (S.D.) in artificial cerebrospinal fluid (1.2 mM Ca²⁺), but decreases at lower Ca²⁺ concentrations in the medium. The presence of quin 2 in the synaptosomes does not affect either the spontaneous release of transmitter (γ-aminobutyric acid) or the release induced by K⁺ depolarisation. In quin 2-loaded synaptosomes, depolarisation by K⁺ causes an abrupt increase in [Ca]_i (less than 2-fold) that is approximately proportional to the extent of depolarisation, whereas depolarisation by veratrine alkaloids produces a slow rise in [Ca]_i. The increase in [Ca]_i produced by K⁺ depolarisation does not occur in the absence of Ca²⁺ in the medium. The data are consistent with a direct correlation between [Ca_i] and transmitter release in functional synaptosomes. The pH in synaptosomes estimated by the indicator quene 1 is 7.04±0.07 and is stable in media containing 5 mM bicarbonate. The pH in synaptosomes was decreased by protoveratrine but not by K⁺ depolarisation.     

Ca++ fluxes in resealed synaptic plasma membrane vesicles

The effect of the monovalent cations Na⁺, Li⁺, and K⁺ on Ca⁺⁺ fluxes has been determined in resealed synaptic plasma membrane vesicle preparations from rat brain. Freshly isolated synaptic membranes, as well as synaptic membranes which were frozen (?80°C), rapidly thawed, and passively loaded with K₂/succinate and ⁴⁵CaCl₂, rapidly released approximately 60% of the intravesicular Ca⁺⁺ when exposed to NaCl or to the Ca⁺⁺ ionophore A 23187. Incubation of these vesicles with LiCl caused a lesser release of Ca⁺⁺. The EC₅₀ for Na⁺ activation of Ca⁺⁺ efflux from the vesicles was approximately 6.6mM. exposure of the Ca⁺⁺-loaded vesicles to 150 mM KCl produced a very rapid (?1 sec) loss of Ca⁺⁺ from the vesicles, but the Na⁺-induced efflux could still be detected above this K⁺ - sensitive effect. Vesicles pre-loaded with NaCl (150 mM) exhibited rapid ⁴⁵Ca uptake with an estimated EC₅₀ for Ca⁺⁺ of 7–10 μM. This Ca⁺⁺ uptake was blocked by dissipation of the Na⁺ gradient. These observations are suggestive of the preservation in these purified frozen synaptic membrane preparations of the basic properties of the Na⁺Ca⁺⁺ exchange process and of a K⁺ - sensitive Ca⁺⁺ flux across the membranes.     

Extracellular potassium controls responsiveness of the noradrenergic cAMP system in the rat retina to fluphenazine

The effects of fluphenazine (FLU) on the noradrenaline (NA) induced cAMP-synthesis in intact rat retinae were studied as a function of extracellular K⁺- and Ca²⁺-ions. Thus NA-induced cAMP levels were measured after incubating intact rat retinae with 50 μM NA in the presence or absence of FLU and in the presence of 1 or 10 mM theophylline. Results were: (1) Experimental condition a: standard NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, at 10 mM theophylline, at 10 μM FLU and at 2 and 0 mM K⁺. FLU does not affect the NA-response at 2 mM K⁺ significantly; however, it inhibits the NA-response at 0 mM K⁺ in this condition. (2) Experimental condition b: NA-responses were measured after incubating retinae at 0.125 mM Ca²⁺, 10 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU replaces a Ca²⁺ function probably connected with the synthesis part of the NA-cAMP system and NA-responses in this low Ca²⁺ condition are consequently enhanced by FLU; however, FLU inhibits the NA-response at 0 mM K⁺ in this condition. (3) Experimental condition c: NA-responses were measured after incubating retinae at 0.75 mM Ca²⁺, 1 mM theophylline, 10 μM FLU and at 2 and 0 mM K⁺. At 2 mM K⁺ FLU enhances the NA-response by further inhibition of the degradation part of the NA-cAMP system; FLU inhibits the NA-response at 0 mM K⁺ in this condition. (4) The inhibitions of the NA-responses by FLU at 0 mM K⁺ in all three conditions a, b and c showed an apparent K_m of 1 μM. (5) Low concentrations of K⁺ (0.4–0.8 mM) maintain the property of FLU to enhance the NA-responses at condition b (0.125 mM Ca²⁺) and at condition c (1 mM theophylline). Results suggest that the activation of NA-receptor coupled adenylate cyclases (NA-AC-ases) by NA, resulting in activation of phosphodiesterase activity by the NA-elevated cAMP-levels, is sustained by (a) membraneous factor(s) connected to the NA-receptor. This (these) factor(s) is (are) switched off in the absence of K⁺. Evidence has been presented, that Ca²⁺ and FLU do not have access to this intramembraneous factor-enzyme activating moiety of the NA-cAMP system at 0 mM K⁺. Between 0.4 and 0.8 mM K⁺ the factor-enzyme-NA-receptor complex is still intact.     

Arachidonic acid release from K+-evoked depolarization of brain synaptosomes

Rat brain synaptosomes prelabeled with [¹⁴C]arachidonate in their phospholipids were superfused with well oxygenated Krebs-Ringer-bicarbonate solution containing 0.2% BSA and subsequently depolarized by elevating the K⁺ concentration in the superfusion medium from 5 to 55 mM. The efflux of labeled arachidonate at steady state was 0.19% (n = 12) of total radioactivity per min. In the presence of 2.5 mM Ca²⁺, high K⁺ (55 mM) in the medium elicited an increase in arachidonate efflux which amounted to 121.4% (n = 6) of control. Both Ca²⁺ and BSA were required for the stimulated efflux of arachidonate during K⁺-depolarization. Under the same condition, K⁺-stimulation also evoked the release of [³H]norepinephrine which was preloaded into the synaptosomes prior to superfusion. EGTA abolished the stimulated release of both arachidonate and norepinephrine during K⁺-depolarization. These results, together with the loss of labeled arachidonic acid from phospholipids (Majewska and Sun, 1982), indicate that deacylation of membrane lipids is involved in synaptic functions.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca²⁺ release by photolysis of caged InsP₃ have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca²⁺-activated Cl⁻ and K⁺ currents. Photolytic release of InsP₃ from caged InsP₃ at 100 Joules caused transient inward (V_H = 60 mV) and outward (V_H = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl⁻ concentration was decreased, and the outward current was suppressed by K⁺ channel blockers, indicating that they were carried by Cl⁻ and K⁺, respectively. Intracellular pre-loading of the InsP₃ receptor antagonist heparin or the Ca²⁺ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca²⁺-dependent Cl⁻ and K⁺ currents underlies the inward and the outward currents. At low flash intensities, InsP₃ caused Ca²⁺ release which normally activated the K⁺ and Cl⁻ currents in a mono-transient manner. At higher intensities, however, InsP₃ induced an additional delayed outward K⁺ current (I_K(delay)). I_K(delay) was independent of the initial K⁺ current, independent of extracellular Ca²⁺, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca²⁺ from caged Ca²⁺ did not mimic the I_K(delay). It is suggested that Ca²⁺ releases from the InsP₃-sensitive pools in an InsP₃ concentration-dependent manner. Low concentrations of InsP₃ induce the transient Ca²⁺-dependent Cl⁻ and K⁺ currents, which reflects the local Ca²⁺ release, whereas high concentrations of InsP₃ induce a delayed Ca²⁺-dependent K⁺ current, which may reflect the Ca²⁺ wave propagation. J. Cell. Physiol. 174:387–397, 1998. © 1998 Wiley-Liss, Inc.