Regional interaction of endoplasmic reticulum Ca2+ signals between soma and dendrites through rapid luminal Ca2+ diffusion.

The endoplasmic reticulum (ER) Ca2+ store plays a key role in integration and conveyance of Ca2+ signals in highly polarized neurons. The interconnected ER network in neurons generates Ca2+ signals in local domains, but the regional interaction is unclear. Here, we show that continuous or repetitive applications of caffeine produced robust Ca2+ release from the ER Ca2+ store in dendritic areas without severe store depletion, but that similar stimuli applied to soma caused rapid store depletion in acutely isolated midbrain dopamine neurons. Partial emptying of the ER Ca2+ store within a dendrite caused a similar level of store depletion in unstimulated dendrites, as well as in soma. Photobleaching and local stimulation experiments revealed that Ca2+ and the dye trapped within the ER diffused rapidly from the soma to dendrites up to 90 microm, which we could resolve, suggesting that the ER network acts as a functional tunnel for rapid Ca2+ transport. These data imply that the ER in soma acts as a Ca2+ reservoir supplying Ca2+ to the dendritic store, and that the dendritic store, hence, is able to respond to Ca2+-mobilizing input signals endurably.

Modulation of delayed rectifier potassium channels by alpha1-adrenergic activation via protein kinase C zeta and p62 in PC12 cells.

When PC12 cells are exposed to nerve growth factor (NGF), they extend neurites and express autonomic ganglion cell properties. We have previously shown that NGF is capable of inducing p62 expression, enabling the formation of the protein kinase C zeta (PKCzeta)-p62-Kvbeta (beta-subunit of delayed rectifier K+ channel) complex, a Kv channel-modulating complex. The formation of this complex results in the shifting of the Kv channel activation curve to the left via PKCzeta activity. During the experiments, we noted that PC12 cells in a high-density culture exhibited a Kv channel activation curve shift similar to that observed in the NGF-treated cells. Therefore, we hypothesized that catecholamines released from PC12 cells may induce p62 expression. In order to test this idea, cells in a low-density culture were treated for 24h with norepinephrine (NE). In these cells, we noted a leftward shift of the activation curve. The presence of the alpha1-adrenergic antagonist specifically prevented the effects of NE. Pre-treatment of the low-density cells with alpha1-agonists induced changes similar to those associated with NE, confirming that NE modulates Kv channels via the alpha1-adrenergic receptor. NE's effects were blocked by treatment with PKCzeta specific inhibitors. Using Western blotting, we observed increased levels of p62 expression in both the high-density cells and the NE-treated low-density cells. These results suggest that locally secreted NE induces an increase in p62 expression, and also exerts a modulatory effect on Kv channels via the PKCzeta-p62-Kvbeta channel modulating complex.

Low affinity cholecystokinin receptor inhibits cholecystokinin- and bombesin-induced oscillations of cytosolic Ca2+ concentration.

We have investigated whether low affinity cholecystokinin (CCK) receptors suppress agonist-induced rises of cytosolic free Ca(2+) concentration ([Ca(2+)]c) in pancreatic acinar cells by using properties of caffeine. A high concentration of caffeine (20 mM) completely blocked inositol 1,4,5-trisphosphate (InsP(3))-induced [Ca(2+)]c rises but spared the InsP(3)-independent long-lasting [Ca(2+)]c oscillations. In the presence of 20 mM caffeine, only high concentrations of CCK, but not bombesin or JMV-180, suppressed the caffeine-resistant CCK or bombesin-induced [Ca(2+)]c oscillations, indicating that low affinity CCK receptors inhibit agonist-induced [Ca(2+)]c oscillations. It could be one of the underlying mechanisms by which low affinity CCK receptors suppress secretion in pancreatic acinar cells.

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells.

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.

Glutathione potentiates cloned rat brain large conductance Ca(2+)-activated K(+) channels (rSlo).

We have investigated the modulation of a cloned rat brain alpha-subunit of large conductance Ca(2+)-activated K(+) channels (rSlo K(+) channels) by glutathione (GSH), a physiological sulfhydryl-specific reducing reagent. The application of GSH to the intracellular side of excised inside-out macroscopic patches of rslo-transfected HEK293 cells reversibly activated the currents. The activation rate constants of the current were increased while the deactivation rate constants were decreased by GSH at all voltages tested without any change in the voltage dependence of the rate constants. GSH induced a leftward shift of the steady state conductance-voltage relationship curve of the current with no change in the slope of the curve. These results suggest that modulation by GSH may constitute an important regulatory mechanism of neuronal large conductance Ca(2+)-activated K(+) channels.

Slow depletion of endoplasmic reticulum Ca(2+) stores and block of store-operated Ca(2+) channels by 2-aminoethoxydiphenyl borate in mouse pancreatic acinar cells.

The compound 2-aminoethoxydiphenyl borate (2-APB) has been used as either a specific membrane-permeable inhibitor for InsP(3) receptors or a store-operated Ca(2+) channel blocker in some cells. In this study, we have investigated actions of 2-APB on Ca(2+) signalling in mouse pancreatic acinar cells by measuring Ca(2+) concentration in the cytosol ([Ca(2+)]c) and in the endoplasmic reticulum (ER). Although 2-APB (50 microM) inhibited or modulated [Ca(2+)]c oscillations generated by a low dose of ACh, it did not block InsP(3)-mediated Ca(2+) release from the ER elicited by high doses of acetylcholine (ACh). 2-APB alone more than 70 microM tended to increase [Ca(2+)]c. When we directly measured Ca(2+) concentration in the lumen of the ER with a low affinity Ca(2+) dye, Mag-fluo-4, 2-APB itself slowly lowered ER Ca(2+) concentration and ACh could further release Ca(2+) from the ER in the presence of 2-APB, suggesting its lack of potency to block InsP(3) receptors. When store-operated Ca(2+) entry was evoked by addition of external Ca(2+) (5 mM) after depletion of Ca(2+) stores, 2-APB (50 microM) substantially blocked the Ca(2+) influx in a reversible manner. We conclude that (a) 2-APB is a good blocker for store-operated Ca(2+) channels, (b) 2-APB could not effectively block InsP(3) receptors, and (c) low doses of 2-APB lower Ca(2+) concentration in the lumen of the ER without a significant elevation of [Ca(2+)]c.

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells.

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.

Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells.

We have investigated the effects of hypoxia and mitochondria inhibitors on the capacitative Ca(2+) entry (CCE) in cultured smooth muscle cells from rabbit small pulmonary arteries. Cyclopiazonic acid (CPA) depleted Ca(2+) from sarcoplasmic reticulum (SR) in Ca(2+)-free medium and subsequent addition of Ca(2+) led to the nifedipine-insensitive, La(3+)-sensitive Ca(2+) influx. The presence of CCE was further verified by the measurement of unidirectional Mn(2+) influx. During the decay phase of the CCE-induced [Ca(2+)]c transients, hypoxia (P(O2) < 50 mmHg) and the mitochondria inhibitor FCCP reversibly increased [Ca(2+)]c, that is La(3+)-sensitive. Once SR is depleted by CPA, subsequent treatment of FCCP slowed the decay of CCE-induced [Ca(2+)]c transients but it did not attenuate Mn(2+) influx. Mitochondrial uptake of incoming Ca(2+) through CCE was demonstrated by additional increase in [Ca(2+)]c with Ca(2+) ionophore after terminating CCE. Together, it is suggested that the augmentation of CCE-induced [Ca(2+)]c transients by hypoxia and FCCP reflects a net gain of [Ca(2+)]c by the inhibition of mitochondrial Ca(2+) uptake.

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells.

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.

Regulation of magnesium-inhibited cation current by actin cytoskeleton rearrangement.

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells.

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.

Inhibitory effect of somatostatin on secretin-induced augmentation of the slowly activating K+ current (IKs) in the rat pancreatic acinar cell.

Recently, the slowly-activating voltage-dependent K+ channel current (IKs) has been reported in the rat pancreatic acinus (RPA). IKs is modulated positively by ACh and secretin, Ca2+ - and cAMP-mediated secretagogues, respectively. In this study, we investigated the effect of somatostatin (SS), a well-known inhibitory hormone of pancreatic fluid secretion, on I(Ks) in RPAs. The whole-cell patch clamp technique was applied to intact RPAs. Step-like depolarizations from -60 mV to above -40 mV induced IKs, a response blocked by the chromanols 293B [concentration for half-maximal inhibition (IC50) 5.3 microM), HMR-1556 (IC50 0.17 microM) and IKs 420 (IC50 2 nM). The application of secretin (5 nM), forskolin (5 microM) or 8-Br-cAMP (0.3 mM) increased the amplitude of IKs two- to fourfold. The addition of SS (1-100 nM) markedly suppressed the augmentation of IKs by secretin or forskolin but had no effect on IKs stimulated by 8-Br-cAMP, nor did SS block the Ca2+ -mediated augmentation of IKs by ACh. These results suggest that the effect of SS on IKs in the rat pancreatic acinar cell is mediated by inhibition of cAMP production, which may play a role in the negative regulation of the exocrine pancreas by SS.

Two different Ca2+-dependent inhibitory mechanisms of spontaneous firing by glutamate in dopamine neurons.

The excitatory neurotransmitter, glutamate, generates a characteristic burst-pause type of firing in midbrain dopamine neurons in association with the reward behavior, but the cellular mechanism by which glutamate generates these bursts is unknown. Here, we show that the bursts in spontaneously firing dopamine neurons can be generated by the combinative actions of the brief stimulatory and the subsequent Ca(2+)-dependent inhibitory signals in response to glutamate stimulation. The two Ca(2+)-dependent firing-extinction signals are activated by different glutamate receptors. Although the activation of metabotropic glutamate receptors rapidly stopped the enhanced firing through the Ca(2+) release from intracellular stores, the activation of NMDA and AMPA/kainate receptors abolished the firing immediately after termination of the stimulation due to the Ca(2+) accumulation in the cell. These two Ca(2+)-dependent inhibitory mechanisms appear to participate in the generation of characteristic bursts in dopamine neurons by controlling the maximum firing number of single bursts and the duration of post-firing pauses.

Membrane-delimited regulation of novel background K+ channels by MgATP in murine immature B cells.

In WEHI-231, a representative immature B cell line, Ca(2+) entry is paradoxically augmented by treatment with 2-aminoethoxydiphenyl borate (2-APB), a blocker of inositol 1,4,5-trisphosphate receptor and of nonselective cation channels (Nam, J. H., Yun, S. S., Kim, T. J., Uhm, D.-Y., and Kim, S. J. (2003) FEBS Lett. 535, 113-118). The initial goal of the present study was to elucidate the effects of 2-APB on membrane currents, which revealed the presence of novel K(+) channels in WEHI-231 cells. Under whole-cell patch clamp conditions, 2-APB induced background K(+) current (I(K,bg)) and hyperpolarization in WEHI-231 cells. Lowering of intracellular MgATP also induced the I(K,bg). The I(K,bg) was blocked by micromolar concentrations of quinidine but not by tetraethylammonium. In a single channel study, two types of voltage-independent K(+) channels were found with large (346 picosiemens) and medium conductance (112 picosiemens), named BK(bg) and MK(bg), respectively. The excision of membrane patches (inside-out (i-o) patches) greatly increased the P(o) of BK(bg). In i-o patches, cytoplasmic MgATP (IC(50) = 0.18 mm) decreased the BK(bg) activity, although non-hydrolyzable adenosine 5'-(beta,gamma-imino)triphosphate had no effect. A pretreatment with Al(3+) or wortmannin (50 microm) blocked the inhibitory effects of MgATP. A direct application of phosphoinositide 4,5-bisphosphate (10 microm) inhibited the BK(bg) activity. Meanwhile, the activity of MK(bg) was unaffected by MgATP. In cell-attached conditions, the BK(bg) activity was largely increased by 2-APB. In i-o patches, however, the MgATP-induced inhibition of BK(bg) was weakly reversed by the addition of 2-APB. In summary, WEHI-231 cells express the unique background K(+) channels. The BK(bg)s are inhibited by membrane-delimited elevation of phosphoinositide 4,5-bisphosphate. The activation of BK(bg) would hyperpolarize the membrane, which augments the calcium influx in WEHI-231 cells.

Glutamate-mediated [Ca2+]c dynamics in spontaneously firing dopamine neurons of the rat substantia nigra pars compacta.

The mechanism by which glutamate regulates the cytosolic free Ca2+ concentration ([Ca2+]c) in spontaneously firing dopamine neurons is not clear. Thus we have investigated the glutamate-mediated [Ca2+]c dynamics in the acutely isolated dopamine neurons from the rat substantia nigra pars compacta by measuring [Ca2+]c and spontaneously occurring action potentials (SAPs). The freshly isolated dopamine neurons showed tetrodotoxin (TTX)-sensitive spontaneous firing of 2-3 Hz and the resting [Ca2+]c decreased with abolition of the SAPs. The level of [Ca2+]c was affected by the spontaneous firing rate. In the presence of the Na+ channel antagonist, TTX (0.5 microM), glutamate increased [Ca2+]c by activating different glutamate receptors depending on the glutamate concentration used. Addition of glutamate at low concentrations (<3 microM) raised [Ca2+]c mainly by activating metabotropic glutamate receptors (mGluR), whereas at high concentrations (>10 microM) it raised [Ca2+]c mainly by activating AMPA/kainate receptors. The contribution of NMDA receptors to the glutamate-mediated [Ca2+]c rises was largest at intermediate concentrations of glutamate. Activation of mGluR elicited a Ca2+ release from intracellular Ca2+ stores and continuous Ca2+ influx out of the cell. The spontaneous firing activities were highly enhanced by submicromolar levels of glutamate and abolished at levels above 10 microM. From these results, we conclude that at low glutamate concentrations the [Ca2+]c in the dopamine neurons is mainly governed by mGluR and the firing activities, whose rate is regulated at submicromolar glutamate concentrations, but at higher glutamate concentrations [Ca2+]c is dominantly affected by AMPA/kainate receptors.

Effects of KCNQ1 channel blocker, 293B, on the acetylcholine-induced Cl- secretion of rat pancreatic acini.

In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl- channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl- secretion, the intracellular Cl- concentration ([Cl-]i) of RPAs was monitored using SPQ, a fluorescent dye quenchable by Cl-, and the effects of K+ channel blockers were examined. Also, the secretion of fluid and enzyme from the whole pancreas of rat was measured. The fluorescence of RPAs loaded with SPQ (FSPQ) was slightly increased by the application of ACh (ACh-Delta FSPQ), indicating net secretion of Cl-. However, the relative change of FSPQ normalized to the control fluorescence (F/F0) of RPAs was only about 20% of the effect observed in rat submandibular gland acinus. The ACh-Delta FSPQ of RPAs was not influenced by the pretreatment with 293B (20 micromol/L), a blocker of KCNQ-type K+ channels. Even the cocktail of K+ channel blockers (10 mmol/L TEA, 3 mmol/L Ba2+, 20 micromol/L 293B) exerted only minute inhibitory effects on ACh-Delta FSPQ in RPAs. In the vascularly perfused rat pancreas, the fluid and enzyme secretion induced by ACh was directly measured. 293B and HMR-1556, both specific blockers of KCNQ1 channel, did not block but even enhanced the secretion of fluid and amylase. These results suggest that the role of KCNQ1 channels may not be essential in the Ca2+-mediated Cl- secretion in rat pancreatic acini.

Ca2+-activated Cl- channel currents in rat ventral prostate epithelial cells.

BACKGROUND: In many epithelial tissues, the Cl(-) efflux via Ca(2+)-activated Cl(-) channels (Cl(Ca)) play a key role for the fluid secretion. To elucidate the mechanism of prostatic fluid secretion, the properties of whole-cell chloride conductance were investigated. MATERIALS AND METHODS: Rat prostate secretory epithelial cells (RPSECs) were isolated by collagenase treatment, and were used for the whole-cell voltage clamp. Both extra- and intracellular monovalent cations were replaced by N-methyl-D-glucamate to record the Cl(-) current selectively. RESULTS: A bath application of Ca(2+)-ionophore, ionomycin (0.2 micro M), increased the membrane conductance with outwardly rectifying voltage-dependence. On step-like depolarization from -60 to +80 mV (500 msec), the ionomycin-induced current showed slowly activating kinetics, a known property of Cl(Ca) current (I(Cl(Ca))) of other tissues. The relative permeability of Cl(Ca) to various anions was calculated from the reversal potentials measured under a total replacement of extracellular Cl(-) with various anions, and the relative order of permeability was SCN(-)>I(-)>Br(-)>Cl(-)>>gluconate. The amplitude of I(Cl(Ca)) was decreased by various anion channel blockers: niflumic acid (100 micro M), DPC (100 micro M), DIDS (1 mM), and NPPB (200 micro M). CONCLUSIONS: RPSECs have Cl(Ca) that may provide Cl(-) efflux pathways for the exocrine secretions of the prostate.

Inhibitory effects of PGE(2) on K(+) currents and Ca(2+) oscillations in rat pancreatic acinar cells.

Prostaglandin E(2) (PGE(2)) inhibits pancreatic enzyme secretion and shows a protective action against pancreatitis. In this study, we tested the effects of PGE(2) on the slowly activating voltage-dependent K(+) channel current ( I(Ks)) and cholecystokinin (CCK)-induced oscillations of cytosolic [Ca(2+)] ([Ca(2+)](i)) in rat pancreatic acini (RPA). I(Ks) in RPA is reportedly augmented by both Ca(2+)- and cAMP-mediated secretagogues. PGE(2) (10(-7) M) decreased the amplitude of I(Ks), an effect that was more prominent following prior stimulation with secretin. The application of the membrane-permeable cAMP analogue 8-Br-cAMP prevented the effect of PGE(2) on I(Ks). The Ca(2+)-mediated augmentation of I(Ks) by ACh was unaffected by pretreatment with PGE(2). Using fura-2 fluorescence ratiometry to assess [Ca(2+)](i), CCK (

K+ channel currents in rat ventral prostate epithelial cells.

BACKGROUND: Electrophysiological function of the normal prostate has not been extensively studied. In particular, ion channel currents and their regulation have not been studied in freshly-isolated prostate cells. METHODS: Rat prostate secretory epithelial (RPSE) cells were isolated by collagenase treatment. Columnar epithelial cells were used for nystatin-perforated, whole-cell voltage clamp, and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. RESULTS: Step-like depolarizing pulses (900 msec) starting from - 90 mV induced outwardly rectifying K(+) currents without inactivation. ACh (10 microM) or ATP (100 microM) increased the outward current and hyperpolarized the cell membrane potential. Ionomycin (0.1 microM), a Ca(2+) ionophore, induced a similar increase in the outward current. TEA (5 mM), charybdotoxin (50 nM), and iberiotoxin (30 nM) inhibited the effect of ACh (or ATP) on the outward current, whereas apamin (100 nM) had no effect. The [Ca(2+)](i) of RPSE cells was increased by ACh, ATP, and UTP. CONCLUSIONS: RPSE cells have iberiotoxin-sensitive Ca(2+)-activated K(+) channels that may play an important role in the exocrine secretions of the prostate.

Purinergic receptors coupled to intracellular Ca2+ signals and exocytosis in rat prostate neuroendocrine cells.

Rat prostate neuroendocrine cells (RPNECs) display a variety of ion channels and exhibit alpha-adrenergic regulation of cytosolic Ca(2+) concentration ([Ca(2+)])(c). In this study, purinergic regulation of [Ca(2+)](c) and exocytosis was investigated in freshly isolated single RPNECs showing chromogranin A immunoreactivity. The presence of P2X and P2Y receptors in RPNECs was verified by the transient activation of Ca(2+)-permeable cationic channels and the release of Ca(2+) from intracellular stores by extracellular ATP, respectively. The transient inward cationic current was effectively activated by alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP) and blocked by 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, suggesting the presence of a P2X(1) or P2X(3) subtype. For the release of stored Ca(2+), ATP and UTP were equally potent, indicating the functional expression of the P2Y(2) or P2Y(4) subtype. The mRNAs for P2X(1) and P2Y(2) were confirmed from reverse transcription-PCR analysis of RPNECs. The application of alpha,beta-MeATP induced large and transient increases in [Ca(2+)](c), which were not attenuated by the blockers of voltage-activated Ca(2+) channels or by depleting intracellular Ca(2+) stores, but were abolished by omitting extracellular Ca(2+). The application of UTP increased [Ca(2+)](c) to 55% of the peak Delta[Ca(2+)](c) induced by alpha,beta-MeATP. The application of alpha,beta-MeATP induced exocytotic responses of RPNECs as monitored by carbon fiber amperometry and capacitance measurements. To our interest, the application of UTP did not induce amperometric currents, but reduced the membrane capacitance, indicating a net endocytosis. From these results, we postulate that a sharp rise in [Ca(2+)](c) by the P2X-mediated Ca(2+) influx is required for exocytosis, whereas the relatively slow release of stored Ca(2+) induces endocytosis in RPNECs.