T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

NTP,the photoproduct of nifedipine,activates caffeine-sensitive ion channels in leech neurons

Leech P neurons possess caffeine-sensitive ion channels in intracellular Ca(2+) stores and in the plasma membrane. The following results indicate that these channels are also activated by 2,6-dimethyl-4-(2-nitrosophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester (NTP), the photoproduct of the L-type Ca(2+) channel-blocker nifedipine: (1) Just like caffeine, NTP evoked Ca(2+) influx and intracellular Ca(2+) release, as well as the influx of various other divalent cations and that of Na(+). (2) In the presence of high NTP or caffeine concentrations the plasma membrane channels close, suggesting desensitization of the channel-activating mechanism. (3) Depending on the concentration, NTP and caffeine induce cross-desensitization or act additively. (4) NTP was effective in the same neurons as caffeine (P, N, Leydig, 101), and it was ineffective in neurons in which caffeine was also ineffective (AP, T, L, 8, AE). (5) In Retzius neurons, NTP and caffeine evoked intracellular Ca(2+) release but no Ca(2+) influx. Despite these parallels, the effects of NTP and caffeine were not identical, which may be due to differences in the mechanisms of channel activation or desensitization and/or to substance-specific side effects. The caffeine-sensitive ion channels were activated by NTP concentrations > or =10 microM, which is almost three orders of magnitude smaller than the threshold concentration of caffeine.     

Calcium-permeable channels in plant cells

Calcium signal transduction is a central mechanism by which plants sense and respond to endogenous and environmental stimuli. Cytosolic Ca(2+) elevation is achieved via two cellular pathways, Ca(2+) influx through Ca(2+) channels in the plasma membrane and Ca(2+) release from intracellular Ca(2+) stores. Because of the significance of Ca(2+) channels in cellular signaling, interaction with the environment and developmental processes in plants, a great deal of effort has been invested in recent years with regard to these important membrane proteins. Because of limited space, in this review we focus on recent findings giving insight into both the molecular identity and physiological function of channels that have been suggested to be responsible for the elevation in cytosolic Ca(2+) level, including cyclic nucleotide gated channels, glutamate receptor homologs, two-pore channels and mechanosensitive Ca(2+) -permeable channels. We provide an overview of the regulation of these Ca(2+) channels and their physiological roles and discuss remaining questions.     

The involvement of voltage-operated calcium channels in somato-dendritic oxytocin release

Magnocellular neurons of the supraoptic nucleus (SON) secrete oxytocin and vasopressin from axon terminals in the neurohypophysis, but they also release large amounts of peptide from their somata and dendrites, and this can be regulated both by activity-dependent Ca(2+) influx and by mobilization of intracellular Ca(2+). This somato-dendritic release can also be primed by agents that mobilise intracellular Ca(2+), meaning that the extent to which it is activity-dependent, is physiologically labile. We investigated the role of different Ca(2+) channels in somato-dendritic release; blocking N-type channels reduced depolarisation-induced oxytocin release from SONs in vitro from adult and post-natal day 8 (PND-8) rats, blocking L-type only had effect in PND-8 rats, while blocking other channel types had no significant effect. When oxytocin release was primed by prior exposure to thapsigargin, both N- and L-type channel blockers reduced release, while P/Q and R-type blockers were ineffective. Using confocal microscopy, we found immunoreactivity for Ca(v)1.2 and 1.3 channel subunits (which both form L-type channels), 2.1 (P/Q type), 2.2 (N-type) and 2.3 (R-type) in the somata and dendrites of both oxytocin and vasopressin neurons, and the intensity of the immunofluorescence signal for different subunits differed between PND-8, adult and lactating rats. Using patch-clamp electrophysiology, the N-type Ca(2+) current density increased after thapsigargin treatment, but did not alter the voltage sensitivity of the channel. These results suggest that the expression, location or availability of N-type Ca(2+) channels is altered when required for high rates of somato-dendritic peptide release.     

A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.     

A constitutive, transient receptor potential-like Ca2+ influx pathway in presynaptic nerve endings independent of voltage-gated Ca2+ channels and Na+/Ca2+ exchange

Calcium levels in the presynaptic nerve terminal are altered by several pathways, including voltage-gated Ca(2+) channels, the Na(+)/Ca(2+) exchanger, Ca(2+)-ATPase, and the mitochondria. The influx pathway for homeostatic control of [Ca(2+)](i) in the nerve terminal has been unclear. One approach to detecting the pathway that maintains internal Ca(2+) is to test for activation of Ca(2+) influx following Ca(2+) depletion. Here, we demonstrate that a constitutive influx pathway for Ca(2+) exists in presynaptic terminals to maintain internal Ca(2+) independent of voltage-gated Ca(2+) channels and Na(+)/Ca(2+) exchange, as measured in intact isolated nerve endings from mouse cortex and in intact varicosities in a neuronal cell line using fluorescence spectroscopy and confocal imaging. The Mg(2+) and lanthanide sensitivity of the influx pathway, in addition to its pharmacological and short hairpin RNA sensitivity, and the results of immunostaining for transient receptor potential (TRP) channels indicate the involvement of TRPC channels, possibly TRPC5 and TRPC1. This constitutive Ca(2+) influx pathway likely serves to maintain synaptic function under widely varying levels of synaptic activity.     

Store-operated Ca2+ entry in porcine airway smooth muscle

Ca(2+) influx triggered by depletion of sarcoplasmic reticulum (SR) Ca(2+) stores [mediated via store-operated Ca(2+) channels (SOCC)] was characterized in enzymatically dissociated porcine airway smooth muscle (ASM) cells. When SR Ca(2+) was depleted by either 5 microM cyclopiazonic acid or 5 mM caffeine in the absence of extracellular Ca(2+), subsequent introduction of extracellular Ca(2+) further elevated [Ca(2+)](i). SOCC was insensitive to 1 microM nifedipine- or KCl-induced changes in membrane potential. However, preexposure of cells to 100 nM-1 mM La(3+) or Ni(2+) inhibited SOCC. Exposure to ACh increased Ca(2+) influx both in the presence and absence of a depleted SR. Inhibition of inositol 1,4,5-trisphosphate (IP)-induced SR Ca(2+) release by 20 microM xestospongin D inhibited SOCC, whereas ACh-induced IP(3) production by 5 microM U-73122 had no effect. Inhibition of Ca(2+) release through ryanodine receptors (RyR) by 100 microM ryanodine also prevented Ca(2+) influx via SOCC. Qualitatively similar characteristics of SOCC-mediated Ca(2+) influx were observed with cyclopiazonic acid- vs. caffeine-induced SR Ca(2+) depletion. These data demonstrate that a Ni(2+)/La(3+)-sensitive Ca(2+) influx via SOCC in porcine ASM cells involves SR Ca(2+) release through both IP(3) and RyR channels. Additional regulation of Ca(2+) influx by agonist may be related to a receptor-operated, noncapacitative mechanism.     

Calcium-induced transitions between the spontaneous miniature outward and the transient outward currents in retinal amacrine cells

Spontaneous miniature outward currents (SMOCs) occur in a subset of retinal amacrine cells at membrane potentials between -60 and -40 mV. At more depolarized potentials, a transient outward current (I(to)) appears and SMOCs disappear. Both SMOCs and the I(to) are K(+) currents carried by BK channels. They both arise from Ca(2+) influx through high voltage-activated (HVA) Ca(2+) channels, which stimulates release of internal Ca(2+) from caffeine- and ryanodine-sensitive stores. An increase in Ca(2+) influx resulted in an increase in SMOC frequency, but also led to a decline in SMOC mean amplitude. This reduction showed a temporal dependence: the effect being greater in the latter part of a voltage step. Thus, Ca(2+) influx, although required to generate SMOCs, also produced a negative modulation of their amplitudes. Increasing Ca(2+) influx also led to a decline in the first latency to SMOC occurrence. A combination of these effects resulted in the disappearance of SMOCs, along with the concomitant appearance of the I(to) at high levels of Ca(2+) influx. Therefore, low levels of Ca(2+) influx, arising from low levels of activation of the HVA Ca(2+) channels, produce randomly occurring SMOCs within the range of -60 to -40 mV. Further depolarization leads to greater activation of the HVA Ca(2+) channels, larger Ca(2+) influx, and the disappearance of discontinuous SMOCs, along with the appearance of the I(to). Based on their characteristics, SMOCs in retinal neurons may function as synaptic noise suppressors at quiescent glutamatergic synapses.     

Survival promotion of mesencephalic dopaminergic neurons by depolarizing concentrations of K+ requires concurrent inactivation of NMDA or AMPA/kainate receptors

The death of dopaminergic neurons that occurs spontaneously in mesencephalic cultures was prevented by depolarizing concentrations of K+ (20-50 mM). However, unlike that observed previously in other neuronal populations of the PNS or CNS, promotion of survival required concurrent blockade of either NMDA or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors by the specific antagonists, MK-801 and GYKI-52466, respectively. Rescued neurons appeared to be healthy and functional because the same treatment also dramatically enhanced their capacity to accumulate dopamine. The effects on survival and uptake were rather specific to dopaminergic neurons, rapidly reversible and still observed when treatment was delayed after plating. Glutamate release increased substantially in the presence of elevated concentrations of K+, and chronic treatment with glutamate induced a loss of dopaminergic neurons that was prevented by MK-801 or GYKI-52466 suggesting that an excitotoxic process interfered with survival when only the depolarizing treatment was applied. The effects of the depolarizing stimulus in the presence of MK-801 were mimicked by BAY K-8644 and abolished by nifedipine, suggesting that neuroprotection resulted from Ca(2+) influx through L-type calcium channels. Measurement of intracellular calcium revealed that MK-801 or GYKI-52466 were required to maintain Ca(2+) levels within a trophic range, thus preventing K+-induced excitotoxic stress and Ca(2+) overload. Altogether, our results suggest that dopaminergic neurons may require a finely tuned interplay between glutamatergic receptors and calcium channels for their development and maturation.     

Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals.

Multiple types of voltage-activated calcium (Ca(2+)) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca(2+) influx through N- and L- type voltage-activated Ca(2+) channels in sympathetic neurons to the depolarization-induced activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine (NE) synthesis, and the depolarization-induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both omega-conotoxin GVIA (1 microM), a specific blocker of N-type channels, and nimodipine (1 microM), a specific blocker of L-type Ca(2+) channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K(+), indicating that Ca(2+) influx through both types of channels contributes to enzyme activation. In contrast, K(+) stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by omega-conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K(+) stimulation of NE release from both ganglia and irises was also blocked completely when omega-conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca(2+) influx through N- and L-type Ca(2+) channels.     

Role of the Na(+)-Ca(2+) exchanger as an alternative trigger of CICR in mammalian cardiac myocytes

Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.     

Excitation-induced Ca(2+) influx in rat soleus and EDL muscle: mechanisms and effects on cellular integrity

In rat skeletal muscle, electrical stimulation increases Ca(2+) influx leading to progressive accumulation of calcium. Excitation-induced Ca(2+) influx in extensor digitorum longus (EDL; fast-twitch fibers) and soleus muscle (slow-twitch fibers) is compared. In EDL and soleus, stimulation at 40 Hz increased (45)Ca uptake 34- and 21-fold and (22)Na uptake 17- and 7-fold, respectively. These differences may be related to the measured 70% higher concentration of Na(+) channels in EDL. Repeated stimulation at 40 Hz elicited a delayed release of lactic acid dehydrogenase (LDH) from EDL (11-fold increase) and soleus (5-fold increase). Continuous stimulation at 1 Hz increased LDH release only from EDL (18-fold). This was associated with increased Ca(2+) content and was augmented at high extracellular Ca(2+) concentration ([Ca(2+)](o)) and suppressed at low [Ca(2+)](o). The data support the hypothesis that excitation-induced Ca(2+) influx is mediated in part by Na(+) channels and that the ensuing increase in intracellular Ca(2+) induces cellular damage. This is most pronounced in EDL, which may account for the repeated observation that prolonged exercise leads to preferential damage to fast-twitch fibers.     

New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.     

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).     

Metabotropic glutamate receptor 5 and calcium signaling in retinal amacrine cells

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca(2+) elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G(q) and G(s). Selective activation of mGluR5 stimulated Ca(2+) elevations that varied in waveform from cell to cell. Experiments using high external K(+) revealed that the mGluR5-dependent Ca(2+) elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca(2+) -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca(2+) elevations is largely due to the influx of extracellular Ca(2+). The sensitivity of the initial phase of the Ca(2+) elevation to thapsigargin indicates that this phase of the response is due to the release of Ca(2+) from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca(2+) elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca(2+) channels and cAMP-gated channels as pathways for Ca(2+) entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca(2+) influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca(2+) release and Ca(2+) influx, possibly through TRP channels.     

Metabotropic Ca2+ channel-induced calcium release in vascular smooth muscle

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca(2+)] owing to either Ca(2+) influx through voltage-gated Ca(2+) channels of the plasmalemma or to receptor-mediated Ca(2+) release from the sarcoplasmic reticulum (SR). Although the ionotropic role of L-type Ca(2+) channels is well known, we review here data suggesting a new role of these channels in arterial myocytes. After sensing membrane depolarization Ca(2+) channels activate G proteins and the phospholipase C/inositol 1,4,5-trisphosphate (InsP(3)) pathway. Ca(2+) released through InsP(3)-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca(2+) signal, thus triggering arterial cerebral vasoconstriction in the absence of extracellular calcium influx. This metabotropic action of L-type Ca(2+) channels, denoted as calcium channel-induced Ca(2+) release, could have implications in cerebral vascular pharmacology and pathophysiology, because it can be suppressed by Ca(2+) channel antagonists and potentiated with small concentrations of extracellular vasoactive agents as ATP.