Depression and anxiety following hematopoietic stem cell transplantation: a prospective population-based study in Germany.

In this prospective multicenter study, we investigated the course of depression and anxiety during hematopoietic stem cell transplantation (HSCT) until 5 years after transplantation adjusting for medical information. Patients were consulted before HSCT (n=239), at 3 months (n=150), 12 months (n=102) and 5 years (n=45) after HSCT. Depression and anxiety were assessed with the Hospital Anxiety and Depression Scale (HADS). Detailed medical and demographic information was collected. Prevalence rates were compared with an age- and gender-matched control group drawn from a large representative sample (n=4110). The risk of depression before HSCT was lower for patients than for the control group (risk ratio (RR), 0.56; 95% confidence interval (CI), 0.39/0.81). Prevalence rates of depression increased from 12 to 30% until 5 years post HSCT. Anxiety rates were most frequently increased before HSCT (29%, RR, 1.31; 95% CI, 1.02/1.68) and then reached a stable level comparable to the background population (RR 0.83, 95% CI, 0.56/1.22). This study confirms the low levels of depression in the short term after HSCT and identifies depression as a long-term effect. Furthermore, it confirms previous results of heightened anxiety before HSCT. Surveillance of symptoms of anxiety during the short-term phase of HSCT and of depression during the following years is crucial.

Investigating the temporal course, relevance and risk factors of fatigue over 5 years: a prospective study among patients receiving allogeneic HSCT.

Although allogeneic hematopoietic stem cell transplantation (HSCT) features severe physical and psychological strain, no previous study has prospectively investigated fatigue beyond 3 years after transplantation. We investigated the temporal course of fatigue over 5 years, compared patients with the general population (GP) and tested for treatment- and complication-related risk factors. Patients were assessed before conditioning (T0, N=239) and at 100-day (T1, N=150), 1-year (T2, N=102) and 5-year (T3, N=45) follow-up. We measured fatigue with the Multidimensional Fatigue Inventory-20. Patients were compared with the GP at T0 and at T3. Global fatigue increased from T0 to T1 (t=3.85, P<0.001), decreased from T1 to T2 (t=-2. 92, P=0.004) and then remained stable (t=0.45, P=0.656). No difference in global fatigue was found between T0 and T3 (t=0.68, P=0.497). Compared with the GP, patients showed higher global fatigue at T0 (t=-6.02, P<0.001) and T3 (t=-2.50, P=0.014). These differences reached meaningful effect sizes (d⩾0.5). Acute and chronic GvHD predicted global fatigue at T1 (γ=0.34, P=0.006) and T2 (γ=0.38, P=0.010), respectively. To conclude, fatigue among allogeneic HSCT patients improves with time, finally returning to pretransplantation levels. However, even after 5 years, the difference from the GP remains relevant. Patients with GvHD are at risk for increased fatigue.

Angiotensin-converting enzyme 2 in acute respiratory distress syndrome.

Angiotensin-converting enzyme (ACE) and ACE2 are highly homologous metalloproteases that provide essential catalytic functions in the renin-angiotensin system (RAS). Angiotensin II is one key effector peptide of the RAS, inducing vasoconstriction and exerting multiple biological functions. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 reduces angiotensin II levels. Accumulating evidence has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Intriguingly, the SARS coronavirus, the cause of severe acute respiratory syndrome (SARS), utilizes ACE2 as an essential receptor for cell fusion and in vivo infections. Moreover, recent studies have demonstrated that ACE2 protects murine lungs from acute lung injury as well as SARS-Spike protein-mediated lung injury, suggesting a dual role of ACE2 in SARS infections and protection from ARDS.

Enhancement of Ca2+-induced Ca2+ release by cyclic ADP-ribose in frog motor nerve terminals.

Ca2+-induced Ca2+ release (CICR) occurs via activation of ryanodine receptors (RyRs) in frog motor nerve terminals after RyRs are primed for activation by repetitive Ca2+ entries, thereby contributing to synaptic plasticity. To clarify how the mechanism of CICR becomes activable by repetitive Ca2+ entries, we studied effects of a RyR modulator, cyclic ADP-ribose (cADPr), on CICR by Ca2+ imaging techniques. Use-dependent binding of fluorescent ryanodine and its blockade by ryanodine revealed the existence of RyRs in the terminals. Repetition of tetani applied to the nerve produced repetitive rises in intracellular Ca2+ ([Ca2+]i) in the terminals. The amplitude of each rise slowly waxed and waned during the course of the stimulation. These slow rises and decays were blocked by ryanodine, indicating the priming, activation and inactivation of CICR. Uncaging of caged-cADPr loaded in the terminals increased the amplitude of short tetanus-induced rises in [Ca2+]i and the amplitude, time to peak and half decay time of the slow waxing and waning rises in [Ca2+]i evoked by repetitive tetani. A cADPr blocker, 8-amino-cADPr, loaded in the terminals decreased the slow waxing and waning component of rises and blocked all the actions of exogenous cADPr. It is concluded that cADPr enhances the priming and activation of CICR. The four-state model for RyRs suggests that cADPr inhibits the inactivation of CICR and increases the activation efficacy of RyR.

HGF/NK4 inhibited VEGF-induced angiogenesis in in vitro cultured endothelial cells and in vivo rabbit model.

AIMS/HYPOTHESIS: As vascular endothelial growth factor (VEGF) plays a pivotal role in the development of diabetic retinopathy, inhibition of angiogenesis induced by VEGF is crucial to treat diabetic retinopathy. HGF (hepatocyte growth factor)/NK4, containing the N-terminal hairpin domain and the four subsequent kringle domains of HGF, is considered as a specific antagonist for HGF. Our aim was to explore the inhibitory effects of HGF/NK4 on angiogenesis induced by VEGF. METHODS: To analyze the in vivo angiogenesis, we used rabbit corneal micropocket assay. Proliferation and migration of human endothelial cells, expression of ets-1, an essential transcription factor for angiogenesis, and the phosphorylation of extracellular signal-regulated kinase (ERK) was examined with or without HGF/NK4. RESULTS: Using corneal micropocket assay, in vivo administration of HGF/NK4 inhibited angiogenesis induced by VEGF. HGF/NK4 inhibited proliferation and migration of human endothelial cells induced by VEGF in a dose-dependent manner. Interestingly, VEGF-mediated phosphorylation of ERK was significantly attenuated by HGF/NK4. Of importance, HGF/NK4 attenuated the increase in ets-1 protein stimulated by VEGF. Nevertheless, HGF/NK4 did not affect phosphorylation of VEGF receptor-2 [kinase domain region (KDR)/foetal liver kinase (Flk)-1]. Although tyrosine phosphatase inhibitor (Na(3)VO(4)), or okadaic acid, serine-threonin kinase inhibitor, did not prevent the inhibition of ERK phosphorylation by HGF/NK4, co-incubation of HGF/NK4 with VEGF significantly diminished mitogen-activated protein (MAP) ERK kinase (MEK) phosphorylation (p<0.01). CONCLUSIONS/INTERPRETATION: Overall, HGF/NK4 inhibited angiogenesis induced by VEGF through inhibition of phosphorylation of ERK and ets-1 expression in in vitro cultured endothelial cells and in vivo rabbit model.

Ca(2+)-dependent Ca(2+) clearance via mitochondrial uptake and plasmalemmal extrusion in frog motor nerve terminals.

Ca(2+) clearance in frog motor nerve terminals was studied by fluorometry of Ca(2+) indicators. Rises in intracellular Ca(2+) ([Ca(2+)](i)) in nerve terminals induced by tetanic nerve stimulation (100 Hz, 100 or 200 stimuli: Ca(2+) transient) reached a peak or plateau within 6-20 stimuli and decayed at least in three phases with the time constants of 82-87 ms (81-85%), a few seconds (11-12%), and several tens of seconds (less than a few percentage). Blocking both Na/Ca exchangers and Ca(2+) pumps at the cell membrane by external Li(+) and high external pH (9.0), respectively, increased the time constants of the initial and second decay components with no change in their magnitudes. By contrast, similar effects by Li(+) alone, but not by high alkaline alone, were seen only on 200 stimuli-induced Ca(2+) transients. Blocking Ca(2+) pumps at Ca(2+) stores by thapsigargin did not affect 100 stimuli-induced Ca(2+) transients but increased the initial decay time constant of 200 stimuli-induced Ca(2+) transients with no change in other parameters. Inhibiting mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenylhydrazone markedly increased the initial and second decay time constants of 100 stimuli-induced Ca(2+) transients and the amplitudes of the second and the slowest components. Plotting the slopes of the decay of 100 stimuli-induced Ca(2+) transients against [Ca(2+)](i) yielded the supralinear [Ca(2+)](i) dependence of Ca(2+) efflux out of the cytosol. Blocking Ca(2+) extrusion or mitochondrial Ca(2+) uptake significantly reduced this [Ca(2+)](i)-dependent Ca(2+) efflux. Thus Ca(2+)-dependent mitochondrial Ca(2+) uptake and plasmalemmal Ca(2+) extrusion clear out a small Ca(2+) load in frog motor nerve terminals, while thapsigargin-sensitive Ca(2+) pump boosts the clearance of a heavy Ca(2+) load. Furthermore, the activity of plasmalemmal Ca(2+) pump and Na/Ca exchanger is complementary to each other with the slight predominance of the latter.

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model.

Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.

Synchronized Ca2+ signals mediated by Ca2+ action potentials in the hippocampal neuron network in vitro.

Periodic, synchronized Ca2+ signals appeared 30-120 min after the application of tetrodotoxin, 4-aminopyridine and Cs+, and became stable in interval (6-47s) for hours. The Ca2+ signals were accompanied by excitatory or inhibitory postsynaptic potentials (excitatory postsynaptic currents (EPSCs) for the former) and blocked by the simultaneous application of 6-cyano-7-nitroquinoxaline-2,3-dione and 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid or treatment with Ca2+ -free solution, nicardipine, or omega-conotoxin MVIIC (omegaCTX), but not with ryanodine, caffeine, thapsigargin or CPP alone. Nicardipine largely, but omegaCTX less, blocked Ca2+ action potentials or voltage pulse-induced Ca2+ currents at the cell soma, while omegaCTX completely blocked autaptic EPSCs. Ca2+ signals within a neuron occurred almost simultaneously in the cell soma and all the processes (> 200 microm), while the latency between Ca2+ signals of neighbouring neurons varied over hundreds of ms like that of Ca2 action potential induction from EPSPs. Ca2+ signals propagated in random directions throughout neural circuits. Thus, when Na+ and K+ channels are blocked, Ca2+ action potentials spontaneously occur somewhere in a neuron, eventually propagate via the cell soma to the presynaptic terminals and activate excitatory synaptic transmission, causing synchronized Ca2+ signals. The results further suggest that the axon of hippocampal neurones have the potential ability to convey coded information via Ca2+ action potentials.

NK4, a four-kringle antagonist of HGF, inhibits spreading and invasion of human pancreatic cancer cells.

Because of the highly aggressive behaviour, i.e. invasive, disseminative and metastatic properties, the outcome for patients with pancreatic cancer is morbid. A better understanding and interference with the malignant behaviour of pancreatic cancer may provide new directions for treatment. We report here the induction of highly motile and invasive properties in human pancreatic cancer cells by hepatocyte growth factor (HGF) and blockage of these properties by NK4, a newly identified antagonist for HGF. In all of eight human pancreatic cancer cell lines we used (AsPC-1, BxPC-3, H-48N, KP-1N, KP-2, KP-3, MIA PaCa-2 and SUIT-2 cells), the c-Met/HGF receptor was expressed at varying levels. Although weak mitogenic activity of HGF was seen only in SUIT-2 and KP-3 cells, HGF strongly stimulated migration and invasion of these pancreatic cancer cells, except for BxPC-3 and MIA PaCa-2 cells. In contrast, migration and invasion potently induced by HGF in KP-1N, KP-3 and SUIT-2 cells were inhibited by NK4. The invasion of SUIT-2 cells was also potently stimulated with the influence of cocultured pancreatic fibroblasts and by ascitic fluid obtained after pancreatic cancer resection, however, invasiveness of the cancer cells in such conditions was practically abolished by NK4. Consistently, the ascitic fluid in patients who had undergone pancreatic cancer surgery contained high levels of HGF. These findings mean that HGF is probably involved in invasion, dissemination, and metastasis of pancreatic cancer, particularly through tumour-stromal interaction and after resection of the pancreatic cancer. NK4, an effective antagonist of HGF, may prove to have the potential for anti-invasion/metastasis.

Vasoactive intestinal peptide-stimulated adenosine 3',5'-cyclic monophosphate formation in cerebral cortex and hypothalamus of chick and rat: comparison of the chicken and mammalian peptide.

Chicken and mammalian (human/porcine/rat) vasoactive intestinal peptides (VIP; 0.01-3 microM), whose structures differ by four amino acid residues in 11, 13, 26 and 28 positions, were compared with respect to their ability to stimulate adenosine 3',5'-cyclic monophosphate (cyclic AMP) formation in the hypothalamus and cerebral cortex of chick and rat. In four tested biological systems, the chicken VIP appeared to be significantly more potent in evoking cyclic AMP response than its mammalian counterpart, the differences were more pronounced in the chick tissues, particularly in the hypothalamus, where the mammalian peptide produced only weak (but significant) effect at the highest used dose, i.e. 3 microM. Pituitary adenylate cyclase-activating polypeptide, a VIP-like peptide, applied as a reference drug at 0.1 microM, strongly stimulated cyclic AMP formation in all tested systems. The data demonstrate significant quantitative differences in biological activity between mammalian and non-mammalian peptides tested in brain tissue of chicks and rats, indicating that usage of the mammalian VIP in at least 'avian' studies may lead to some false conclusions.

HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice.

We reported that NK4, composed of the N-terminal hairpin and subsequent four kringle domains of hepatocyte growth factor (HGF), acts as the competitive antagonist for HGF. We now provide the first evidence that NK4 inhibits tumor growth and metastasis as an angiogenesis inhibitor as well as an HGF antagonist. Administration of NK4 suppressed primary tumor growth and lung metastasis of Lewis lung carcinoma and Jyg-MC(A) mammary carcinoma s.c. implanted into mice, although neither HGF nor NK4 affected proliferation and survival of these tumor cells in vitro. NK4 treatment resulted in a remarkable decrease in microvessel density and an increase of apoptotic tumor cells in primary tumors, which suggests that the inhibition of primary tumor growth by NK4 may be achieved by suppression of tumor angiogenesis. In vivo, NK4 inhibited angiogenesis in chick chorioallantoic membranes and in rabbit corneal neovascularization induced by basic fibroblast growth factor (bFGF). In vitro, NK4 inhibited growth and migration of human microvascular endothelial cells induced by bFGF and vascular endothelial growth factor (VEGF) as well as by HGF. HGF and VEGF activated the Met/HGF receptor and the KDR/VEGF receptor, respectively, whereas NK4 inhibited HGF-induced Met tyrosine phosphorylation but not VEGF-induced KDR phosphorylation. NK4 inhibited HGF-induced ERK1/2 (p44/42 mitogen-activated protein kinase) activation, but allowed for bFGF- and VEGF-induced ERK1/2 activation. These results indicate that NK4 is an angiogenesis inhibitor as well as an HGF antagonist, and that the antiangiogenic action of NK4 is independent of its activity as HGF antagonist. The bifunctional properties of NK4 to act as an angiogenesis inhibitor and as an HGF antagonist raises the possibility that NK4 may prove therapeutic for cancer patients.

Functional triads consisting of ryanodine receptors, Ca(2+) channels, and Ca(2+)-activated K(+) channels in bullfrog sympathetic neurons. Plastic modulation of action potential.

Fluorescent ryanodine revealed the distribution of ryanodine receptors in the submembrane cytoplasm (less than a few micrometers) of cultured bullfrog sympathetic ganglion cells. Rises in cytosolic Ca(2+) ([Ca(2+)](i)) elicited by single or repetitive action potentials (APs) propagated at a high speed (150 microm/s) in constant amplitude and rate of rise in the cytoplasm bearing ryanodine receptors, and then in the slower, waning manner in the deeper region. Ryanodine (10 microM), a ryanodine receptor blocker (and/or a half opener), or thapsigargin (1-2 microM), a Ca(2+)-pump blocker, or omega-conotoxin GVIA (omega-CgTx, 1 microM), a N-type Ca(2+) channel blocker, blocked the fast propagation, but did not affect the slower spread. Ca(2+) entry thus triggered the regenerative activation of Ca(2+)-induced Ca(2+) release (CICR) in the submembrane region, followed by buffered Ca(2+) diffusion in the deeper cytoplasm. Computer simulation assuming Ca(2+) release in the submembrane region reproduced the Ca(2+) dynamics. Ryanodine or thapsigargin decreased the rate of spike repolarization of an AP to 80%, but not in the presence of iberiotoxin (IbTx, 100 nM), a BK-type Ca(2+)-activated K(+) channel blocker, or omega-CgTx, both of which decreased the rate to 50%. The spike repolarization rate and the amplitude of a single AP-induced rise in [Ca(2+)](i) gradually decreased to a plateau during repetition of APs at 50 Hz, but reduced less in the presence of ryanodine or thapsigargin. The amplitude of each of the [Ca(2+)](i) rise correlated well with the reduction in the IbTx-sensitive component of spike repolarization. The apamin-sensitive SK-type Ca(2+)-activated K(+) current, underlying the afterhyperpolarization of APs, increased during repetitive APs, decayed faster than the accompanying rise in [Ca(2+)](i), and was suppressed by CICR blockers. Thus, ryanodine receptors form a functional triad with N-type Ca(2+) channels and BK channels, and a loose coupling with SK channels in bullfrog sympathetic neurons, plastically modulating AP.

Ryanodine- and thapsigargin-insensitive Ca2+-induced Ca2+ release is primed by lowering external Ca2+ in rabbit autonomic neurons.

Rises in cytosolic Ca2+ induced by a high K+ concentration (30 or 60 mM) (K+-induced Ca2+ transient) were recorded by fluorimetry of Ca2+ indicators in cultured rabbit otic ganglion cells. When external Ca2+ ([Ca2+]o) was reduced to a micromolar (10-40 microM) or nanomolar (<10 nM) level prior to high-K+ treatment, K+-induced Ca2+ transients of considerable amplitude (50% of control) were generated in most cells, although those initiated at normal [Ca2+]o were reduced markedly or abolished by reducing [Ca2+]o during exposure to a high K+ concentration. Lowering [Ca2+]o alone occasionally caused a transient rise in cytosolic Ca2+. K+-induced Ca2+ transients at micromolar [Ca2+]o were repeatedly generated and propagated inwardly at a speed slower than that at normal [Ca2+]o, while those at nanomolar [Ca2+]o occurred only once. K+-induced Ca2+ transients at micromolar [Ca2+]o were not blocked by ryanodine (10 microM), carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 5 microM: at 20-22 degrees C but blocked at 31-34 degrees C) or thapsigargin (1-2 microM), but were blocked by Ni2+ (1 mM) or nicardipine (10 microM). Thus, there is a ryanodine-insensitive Ca2+-release mechanism in FCCP- and thapsigargin-insensitive Ca2+ stores in rabbit otic ganglion cells, which is primed by lowering [Ca2+]o and then activated by depolarization-induced Ca2+ entry. This Ca2+-induced Ca2+ release may operate when [Ca2+]o is decreased by intense neuronal activity.

Ca2+ dynamics at the frog motor nerve terminal.

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.

Effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on cyclic AMP formation in the duck and goose brain.

Two molecular forms of pituitary adenylate cyclase-activating polypeptide (PACAP), i.e., PACAP27 and PACAP38 (0.0001-1 microM), as well as vasoactive intestinal polypeptide (VIP; 0.1-3 microM), have been studied for their effects on cyclic AMP formation in the hypothalamus and cerebral cortex of duck and goose. All three peptides concentration-dependently stimulated cyclic AMP production in the tested brain regions of 2-3-weeks-old (young) ducks, with VIP showing at least one order of magnitude weaker activity than PACAP. This characteristics suggests the existence in the duck's brain of adenylyl cyclase-linked PAC1 receptors. Both forms of PACAP also stimulated the nucleotide formation in the cerebral cortex and hypothalamus of 5-6-months-old (adult) ducks or geese grown under natural environment. The peptides-evoked effects in adult and young ducks were comparable, and clearly greater than those found in adult geese. The present data extend our recent observations made on chicks, and suggest PACAP to be a potent stimulator of the cyclic AMP generation in the avian central nervous system.

Modes of propagation of Ca(2+)-induced Ca2+ release in bullfrog sympathetic ganglion cells.

How depolarization-induced Ca2+ entry or caffeine activates Ca(2+)-induced Ca2+ release (CICR) in the cytoplasm and nucleoplasm was studied by recording intracellular Ca2+ ([Ca2+]i) with a confocal microscope in cultured bullfrog sympathetic ganglion cells. The amplitude and propagation speed of voltage pulse-induced rises in [Ca2+]i were greater in the submembrane (< 5 microns depth) region than in the core region, and delayed and smaller, but significant, in the nucleus. Ryanodine and dantrolene reduced the rises in [Ca2+]i in both the cytoplasm and nucleus. A rapid application of high K+ solution induced global rises in [Ca2+]i in both the cytoplasm and nucleoplasm, which were decreased by dantrolene. Caffeine produced a slow, small rise in [Ca2+]i which grew into a global, regenerative rise both in the cytoplasm and nucleoplasm with some inward gradient in the cytoplasm. Each of the high [Ca2+]i phases during caffeine-induced [Ca2+]i oscillation began in the submembrane region, while low [Ca2+]i phases started in the core region. These results suggest that CICR activated by Ca2+ entry or caffeine occurs predominantly in the submembrane region causing an inwardly spreading Ca2+ wave or [Ca2+]i oscillations, and that the nuclear envelope can cause CICR in the nucleoplasm, which is delayed due to Ca2+ diffusion barrier at the nuclear pores.

Functional coupling of Ca(2+) channels to ryanodine receptors at presynaptic terminals. Amplification of exocytosis and plasticity.

Ca(2+)-induced Ca(2+) release (CICR) enhances a variety of cellular Ca(2+) signaling and functions. How CICR affects impulse-evoked transmitter release is unknown. At frog motor nerve terminals, repetitive Ca(2+) entries slowly prime and subsequently activate the mechanism of CICR via ryanodine receptors and asynchronous exocytosis of transmitters. Further Ca(2+) entry inactivates the CICR mechanism and the absence of Ca(2+) entry for >1 min results in its slow depriming. We now report here that the activation of this unique CICR markedly enhances impulse-evoked exocytosis of transmitter. The conditioning nerve stimulation (10-20 Hz, 2-10 min) that primes the CICR mechanism produced the marked enhancement of the amplitude and quantal content of end-plate potentials (EPPs) that decayed double exponentially with time constants of 1.85 and 10 min. The enhancement was blocked by inhibitors of ryanodine receptors and was accompanied by a slight prolongation of the peak times of EPP and the end-plate currents estimated from deconvolution of EPP. The conditioning nerve stimulation also enhanced single impulse- and tetanus-induced rises in intracellular Ca(2+) in the terminals with little change in time course. There was no change in the rate of growth of the amplitudes of EPPs in a short train after the conditioning stimulation. On the other hand, the augmentation and potentiation of EPP were enhanced, and then decreased in parallel with changes in intraterminal Ca(2+) during repetition of tetani. The results suggest that ryanodine receptors exist close to voltage-gated Ca(2+) channels in the presynaptic terminals and amplify the impulse-evoked exocytosis and its plasticity via CICR after Ca(2+)-dependent priming.

Activity-dependent enhancement of miniature excitatory postsynaptic current amplitude and its modulation by protein kinase C in cultured rat sympathetic neurons.

A high K+ solution increased the frequency of miniature excitatory postsynaptic currents (MEPSCs) and intracellular Ca2+ concentration ([Ca2+]i) in cultured rat sympathetic neurons. Repetition or continuation of high K+ treatment increased MEPSC amplitude, acetylcholine-induced currents and the averaged rise in [Ca2+]i per single MEPSC. The enhancement of MEPSCs lasted over 30 min and was inhibited by intracellular BAPTA and phorbol ester, but not by atropine. The results suggest that repeated Ca2+ entry through the channel pore of nicotinic acetylcholine receptor enhances the efficacy of its opening and the activation of protein kinase C inhibits the enhancement.