Voltage-sensitive Ca Channels Research Articles

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein‐mediated constitutive inhibition of calcium channels

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Long-term alteration of gamma-aminobutyric acid B receptor subunits in immature rats after recurrent febrile seizures

Febrile seizure (FS) is closely related to an altered transmission of gamma-aminobutyric acid (GABA). GABA exerts its effects through ionotropic receptors (GABA(AR) and GABA(CR)) and metabotropic receptors (GABA(BR)). GABA(BRs) are located at pre- and postsynaptic sites. Stimulation of postsynaptic receptors generates long-lasting inhibitory postsynaptic potentials (IPSPs) that are important for the fine-tuning of inhibitory neurotransmission and caused by an increase in K(+) conductance. At presynaptic sites, GABA(BRs) mediate a suppression on the release of neurotransmitters such as of GABA or glutamate by inhibiting voltage-sensitive Ca(2+) channels. The present study aimed to explore the long-term changes of GABA(B) receptor subunits in immature rats after recurrent febrile seizures. Rats were randomly divided into control group and hyperthermia treatment group. The control rats (n = 64) were put into 37 degrees C water for 5 minutes. Rats with hyperthermia treatment were put into 44.8 degrees C water for 5 minutes. If a rat in hyperthermia treatment group showed seizure within 5 min, the rat was taken out of the water as soon as the seizure occurred. Water-immersion was carried out 10 times, once every 2 days. Rats showing 10 seizures (FS(10), n = 64) were studied. Rats exposed to hyperthermia for 10 times without seizure were also studied as hyperthermia-only (H, n = 64) group. Rats showing one seizure at the last time of 10 times of hyperthermia treatment were studied as one-seizure group (FS(1), n = 64). The other rats were studied for other research. The changes of GABA(B)R(1) and GABA(B)R(2) co-localization were detected by double fluorescence;the quantitative alteration of GABA(B)R(1) and GABA(B)R(2) were detected by quantitative RT-PCR; the binding of GABA(B)R(2) to GABA(B)R(1) was detected by immunoprecipitation/Western blot. GABA(B)R(1), GABA(B)R(2), and the binding of GABA(B)R(2) to GABA(B)R(1) decreased after the last febrile seizure in FS(10) group, the expression of GABA(B)R(1) returned to normal in later phase while GABA(B)R(2) and the binding of them did not. Recurrent FS down-regulated the expression of GABA(B)R subunits in a long term.

β-Adrenoceptors and potassium channels

The existence of two subtypes of adrenergic receptors, the α-adrenoceptors (αAR) and the β-adrenoceptors (βAR), was first demonstrated by Ahlquist (1948), and since then they have been extensively studied and characterised. They are receptors for the endogenous catecholamines epinephrine and norepinephrine and have a very wide tissue distribution (Table 1). In the case of βAR, Lands et al. (1967) found evidence for the existence of two types of βAR β1 and β2—by measuring the relative potency of sympathomimetic amines in different tissues. Subsequently, Emorine et al. (1989) isolated and cloned a third βARsubtype—the β3 adrenoceptor. Pharmacological evidence suggested a fourth type of βAR (Kaumann 1997; Molenaar et al. 1997; Galitzky et al. 1998), but it has not been cloned. In fact, recent evidence suggests that the putative β4AR, identified in the heart, is probably a low-affinity state of the β1AR (Granneman 2001; Kaumann et al. 2001; Sarsero et al. 2003; Joseph et al. 2004a,b; Arch 2004). All βAR are G-protein-coupled receptors, the predominant signal transduction mechanism of which is coupling of a Gs protein to adenylyl cyclase (AC), which becomes activated and catalyses the conversion of adenosine triphosphate (ATP) to cyclic 3′,5′ adenosine monophosphate (cAMP). cAMP activates protein kinase A (PKA) by binding to its regulatory subunit, causing PKA to dissociate from the catalytic subunit, thereby rendering it active. PKA is a serine/threonine protein kinase that targets a number of intracellular proteins, thereby eliciting a series of specific cellular responses. Such is the well-known “classical” mechanism for βAR signal-transduction. In the last two decades, however, it has become clear that this is by no means the only mechanism by which βAR give rise to intracellular responses. For example, in the heart, β2AR can couple to both Gs and Gi proteins. Coupling to Gs activates the cAMP-PKA pathway, mediating positive inotropic effects in the same manner as β1AR; by contrast, coupling to the Gi protein modifies the outcome of Gs signalling through activation of the PI3 kinase-Akt pathway (Zheng et al. 2005). In rat aorta, β2AR give rise to stimulation of nitric oxide biosynthesis, partially through PKA signalling but partly also through PI3 kinase-Akt activation (Ferro et al. 2004). In skeletal and cardiac muscle cells, Gs can directly enhance the activation of plasmalemmal voltage-sensitive Ca channels (Brown and Birnbaumer 1988), and this represents another potential mechanism of βAR signalling which is Gs-mediated but cAMP-PKA-independent. Interestingly, the coupling to voltage-sensitive Ca channels may differ between β1AR and β2AR (Foerster et al. 2004) In more recent years, it has become evident that, in a wide variety of tissues, βAR can also cause activation of potassium channels to give rise to their cellular effects; and interestingly, the potassium channels involved appear to vary widely from one tissue to another, for no obvious reason. In conscious dogs, β2AR-mediated dilatation of resistance coronary vessels involves both the opening of KATP channels and nitric oxide formation, and inhibition of nitric oxide synthase was found to antagonise lemakalim responses (Ming et al. 1997). In isolated canine saphenous veins and rat mesenteric arteries, the βAR agonist Naunyn-Schmiedeberg’s Arch Pharmacol (2006) 373:183–185 DOI 10.1007/s00210-006-0065-2

Effect of Aβ exposure on the mRNA expression patterns of voltage-sensitive calcium channel α 1 subunits (α 1A–α 1D) in human SK-N-SH neuroblastoma

Evidence for loss of Ca 2+ homeostasis through voltage-sensitive Ca 2+ channels (VSCCs) contribution to neuronal degeneration induced by β-amyloid protein (Aβ) is considerable and rapidly increasing. Thus, the expression patterns of four α 1 subunits for P/Q (α 1A)-, N (α 1B)-, and L (α 1C and α 1D)-type VSCCs before and after Aβ exposure were investigated in human SK-N-SH neuroblastoma. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a constitutive and abundant co-expression of mRNA for α 1A and α 1D subunit in control cells. The mRNA expression of another L-type subunit α 1C was undetectable in control cells while N-type subunit α 1B was relative lower when compared to α 1A and α 1D subunits. Interestingly, mRNA levels of α 1A, α 1B, and α 1C were remarkably and time-dependently increased in response to Aβ (20 μM) for 72 h culture period. In contrast, the constitutively expressed α 1D mRNA was not further modified during Aβ exposure. Western blot analysis of four α 1 subunits expression was consistent with the findings obtained by RT-PCR. In conclusion, our results suggested that P/Q-, N-, as well as L-type Ca 2+ channel genes might be existed in SK-N-SH cells. Among them, mRNA for α 1A, α 1B, and α 1D were expressed constitutively while α 1C were inducible. Furthermore, Aβ exposure selectively modulates the transcription of α 1A, α 1B, and α 1C subunits. These suggested that except activating of existed VSCCs, up-regulation of α 1 subunits expression might also contribute to Aβ-induced neuronal toxicity and the complex of these VSCCs expression may participate in Ca 2+ current disturbance in Alzheimer's disease.

Novel Ca 2+ signaling pathway for AVP mediated vasoconstriction

[Arg8]-vasopressin (AVP) is a peptide hormone which exerts vasoconstrictor effects by binding to V1a vasopressin receptors on vascular smooth muscle cells (VSMC). This effect is attributed to activation of phosphoinositidase C (PIC) and consequent release of Ca2+ from the sarcoplasmic reticulum. Interestingly, half-maximal activation of PIC requires nanomolar AVP concentrations, whereas circulating AVP concentrations rarely exceed 100pM. Using cultured VSMC, we previously identified a novel Ca2+ signaling pathway activated by 10–100pM AVP. This Ca2+ signaling pathway is distinguished from the PIC pathway by its dependence upon protein kinase C (PKC) activation and L-type voltage-sensitive Ca2+ channels (VSCC). In the present study, we tested the hypothesis that picomolar AVP concentrations would induce PKC- and VSCC-dependent vasoconstriction in isolated pressurized rat mesenteric arteries. AVP (10−14 to 10−6 M) induced a concentration-dependent constriction of arteries that was reversible with a V1a vasopressin receptor antagonist. 30pM AVP induced significant vasoconstriction (~45% of maximum), which was prevented by PKC inhibition with calphostin-C (250nM) or Ro-31–8220 (1μM) as well as by blockade of VSCC with verapamil (10μM). In contrast, maximal vasoconstriction induced by 10nM AVP was insensitive to either PKC inhibition or blockade of VSCC. These results suggest that two dose-dependent signaling pathways exist for AVP-induced vasoconstriction. Accordingly, we conclude that the vasoconstrictor actions of picomolar AVP within the systemic circulation may be dependent upon the novel Ca2+ signaling pathway involving PKC activation and L-type VSCC. NIH RO1-HL070670 and Falk Fellowship

Osteoblast Ca2+ permeability and voltage-sensitive Ca2+ channel expression is temporally regulated by 1,25-dihydroxyvitamin D3

The cardiac subtype of the L-type voltage-sensitive Ca(2+) channel (VSCC) Cav1.2 (alpha(1C)) is the primary voltage-sensitive channel responsible for Ca(2+) influx into actively proliferating osteoblasts. This channel also serves as the major transducer of Ca(2+) signals in growth-phase osteoblasts in response to hormone treatment. In this study, we have demonstrated that 24-h treatment of MC3T3-E1 preosteoblasts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], a coupling factor for bone resorption, coordinately downregulates Cav1.2 (alpha(1C)) and uniquely upregulates T-type channel Cav3.2 (alpha(1H)). No other voltage-sensitive channel alpha-subunit of the 10 that were surveyed was upregulated by 1,25(OH)(2)D(3). The shift from predominantly L-type to T-type channel expression has been demonstrated to occur at both mRNA and protein levels detected using quantitative PCR and immunohistochemistry with antibodies specific for each channel type. Functional and pharmacological studies using specific inhibitors have revealed that treatment with 1,25(OH)(2)D(3) also alters the Ca(2+) permeability properties of the osteoblast membrane from a state of primarily L-current sensitivity to T-current sensitivity. We conclude that the L-type channel is likely to support proliferation of osteoblast cells, whereas T-type channels are more likely to be involved in supporting differentiated functions after 1,25(OH)(2)D(3)-mediated reversal of remodeling has occurred. This latter observation is consistent with the unique expression of the T-type VSCC Cav3.2 (alpha(1H)) in terminally differentiated osteocytes as we recently reported.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic Adenosine 3′,5′-Monophosphate and Ca2+/Calmodulin-Dependent Signaling Pathways in PACAP-Induced GH Secretion and GH Gene Expression in Grass Carp Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca(2+) entry through voltage-sensitive Ca(2+) channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca(2+) entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca(2+) or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC(1) receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca(2+) entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.

General mechanisms of axonal damage and its prevention

Axonal degeneration is a prominent pathological feature in multiple sclerosis observed over a century ago. The gradual loss of axons is thought to underlie irreversible clinical deficits in this disease. The precise mechanisms of axonopathy are poorly understood, but likely involve excess accumulation of Ca ions. In healthy fibers, ATP-dependent pumps support homeostasis of ionic gradients. When energy supply is limited, either due to inadequate delivery (e.g., ischemia, mitochondrial dysfunction) and/or excessive utilization (e.g., conduction along demyelinated axons), ion gradients break down, unleashing a variety of aberrant cascades, ultimately leading to Ca overload. During Na pump dysfunction, Na can enter axons through non-inactivating Na channels, promoting axonal Na overload and depolarization by allowing K egress. This will gate voltage-sensitive Ca channels and stimulate reverse Na–Ca exchange, leading to further Ca entry. Energy failure will also promote Ca release from intracellular stores. Neurotransmitters such as glutamate can be released by reverse operation of Na-dependent transporters, in turn activating a variety of ionotropic and metabotropic receptors, further exacerbating overload of cellular Ca. Together, this Ca overload will inappropriately stimulate a variety of Ca-dependent enzyme systems (e.g., calpains, phospholipases), leading to structural and functional axonal injury. Pharmacological interruption at key points in these interrelated injury cascades (e.g., at voltage-gated Na channels or AMPA receptors) may confer significant neuroprotection to compromised central axons and supporting glia. Such agents may represent attractive adjuncts to currently available immunomodulatory therapies.

The role of calmodulin as a signal integrator for synaptic plasticity

Excitatory synapses in the brain show several forms of synaptic plasticity, including long-term potentiation (LTP) and long-term depression (LTD), which are initiated by increases in intracellular Ca(2+) that are generated through NMDA (N-methyl-D-aspartate) receptors or voltage-sensitive Ca(2+) channels. LTP depends on the coordinated regulation of an ensemble of enzymes, including Ca(2+)/calmodulin-dependent protein kinase II, adenylyl cyclase 1 and 8, and calcineurin, all of which are stimulated by calmodulin, a Ca(2+)-binding protein. In this review, we discuss the hypothesis that calmodulin is a central integrator of synaptic plasticity and that its unique regulatory properties allow the integration of several forms of signal transduction that are required for LTP and LTD.

Raloxifene Relaxes Rat Pulmonary Arteries and Veins: Roles of Gender, Endothelium, and Antagonism of Ca2+ Influx

Effects of raloxifene have been documented in the systemic circulation. However, its impact on the pulmonary circulation is unclear. The present study investigated the role of gender, endothelial modulation, and Ca(2+) channel in relaxations evoked by raloxifene in rat pulmonary arteries and veins. Vascular responses were studied on isolated pulmonary blood vessels mounted in a myograph and constricted by U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha)). Constrictions to CaCl(2) were studied in Ca(2+)-free, 60 mM K(+) solution. Changes in the intracellular calcium ion concentration ([Ca(2+)](i)) in vascular smooth muscle were measured using a calcium fluorescence imaging method. Raloxifene was more effective in relaxing U46619-constricted pulmonary arteries from male than female rats. Raloxifene-induced relaxation was unaffected by ICI 182,780 [7alpha-[9-[(4,4,5,5,5,-pentafluoropentyl)-sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol], inhibition of the nitric oxide (NO) pathway, or removal of the endothelium. In arteries without endothelium, raloxifene attenuated CaCl(2)-induced constriction and CaCl(2)-stimulated increase in [Ca(2+)](i) with similar potencies. Raloxifene caused endothelium-independent relaxations in pulmonary veins, albeit to a lesser degree than in pulmonary arteries. The venous responses showed a gender difference because raloxifene was more potent in male veins. In summary, raloxifene relaxed rat pulmonary arteries, and this effect did not involve the endothelium/NO or ICI 182,780-sensitive estrogen receptors. Raloxifene, like nifedipine, reduced constriction and [Ca(2+)](i) increase in response to CaCl(2) in high K(+) solution. Raloxifene also relaxed high K(+)-constricted pulmonary veins. Our data indicate that raloxifene acutely relaxes rat pulmonary blood vessels primarily via inhibition of Ca(2+) influx through voltage-sensitive Ca(2+) channels. Finally, raloxifene induced more relaxation in blood vessels isolated from male than female rats.

State-Dependent Calcium Signaling in Dendritic Spines of Striatal Medium Spiny Neurons

Striatal medium spiny neurons (MSNs) in vivo undergo large membrane depolarizations known as state transitions. Calcium (Ca) entry into MSNs triggers diverse downstream cellular processes. However, little is known about Ca signals in MSN dendrites and spines and how state transitions influence these signals. Here, we develop a novel approach, combining 2-photon Ca imaging and 2-photon glutamate uncaging, to examine how voltage-sensitive Ca channels (VSCCs) and ionotropic glutamate receptors contribute to Ca signals in MSNs. We find that upstate transitions switch the VSCCs available in dendrites and spines, decreasing T-type while enhancing L-type channels. Moreover, these transitions change the dominant synaptic Ca source from Ca-permeable AMPA receptors to NMDA receptors. Finally, pairing bAPs with synaptic inputs generates additional synaptic Ca signals due to enhanced Ca influx through NMDA receptors. By altering the sources, amplitude, and kinetics of spine Ca signals, state transitions may gate synaptic plasticity and gene expression in MSNs.

Determination of exocytosis mechanisms of DOPA in rat striatum using in vivo microdialysis

To explore the exocytosis mechanism of striatal 3,4-dihydroxyphenylalanine (DOPA), this study determined the interaction between voltage-sensitive Ca 2+-channel (VSCC) and SNARE on releases of DOPA and glutamate in rat striatum using microdialysis. Inhibitors of VSCCs and SNAREs did not affect basal glutamate release but decreased basal DOPA release, however, blocking effects of P-type-VSCC and synaptobrevin inhibitors were weaker than those of N-type-VSCC and syntaxin. The K +-evoked releases of DOPA and glutamate were reduced by inhibitors of P-type-VSCC and synaptobrevin predominantly and by inhibitors of N-type-VSCC and syntaxin weakly. However, interaction study between VSCC and SNARE on K +-evoked DOPA release indicates that DOPA release is regulated by different exocytosis mechanism from glutamate and monoamine during the depolarization stage (N-type-VSCC/P-type-VSCC/synaptobrevin and/or combination with N-type-VSCC/synaptobrevin and P-type-VSCC/synaptobrevin). Therefore we conclude that striatal DOPA release might be regulated by its specific exocytosis mechanism via different from dopaminergic presynaptic vesicle.

Integrating rapid responses to 1,25-dihydroxyvitamin D 3 with transcriptional changes in osteoblasts: Ca 2+ regulated pathways to the nucleus

It is well established that 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) treatment of target cells including osteoblasts activates both membrane-initiated rapid Ca 2+ responses linked to influx through voltage sensitive Ca 2+ channels (VSCCs) and longer term nuclear receptor-mediated changes in gene expression. We recently reported use of a cDNA microarray strategy to identify transcriptional changes after 3 and 24 h of treatment with 1,25(OH) 2D 3 and with an analog of 1,25(OH) 2D 3 (25(OH)-16ene-23yne-D 3 [AT]) that activates Ca 2+ influx without binding to the nuclear receptor. Among 5000 different clones on the array filters, we identified families of genes in osteoblasts that were altered two-fold or greater following treatment with 1,25(OH) 2D 3 or analog AT for 3 h. Cluster analysis further revealed complex patterns of changes in gene expression, indicative of multiple pathways to the nucleus. Evidenced by changes in target gene expression, activation of a Ca 2+/CaMK/CREB/CRE pathway clearly occurs and modulates expression of a variety of genes associated with changes in protein secretion including those involved in paracrine regulation of bone resorption, RANKL and osteoprotegerin (OPG). The changes in gene expression can be inhibited by L-type VSCC channel blockers, confirming the role of Ca 2+ entry in pathway activation. These findings provide clear evidence of rapid changes in gene expression associated with Ca 2+ influx after treatment with 1,25(OH) 2D 3, and open the door to novel nuclear receptor-independent signaling pathways that affect gene transcription.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings

It was previously reported that the K +-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or d-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or d-serine on the K +-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K +-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca 2+-free medium and with voltage-sensitive Ca 2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30–50%) dependent on external Ca 2+ and Ca 2+ channel activation. In contrast, the alkalinization-evoked release of [ 3 H ]noradrenaline was highly sensitive to external Ca 2+ removal and to blockade of Ca 2+ channels with ω-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca 2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.

Presynaptic α 2-receptors regulate reverse Na +/Ca 2+-exchange and transmitter release in Na +-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [ 3 H ]noradrenaline ([ 3 H ]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3×10 −5 M; corticosterone, 5×10 −5 M). Substitution of most of the external Na + by Li + (113 mM; [Na +] 0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [ 3 H ]NA in a tetrodotoxin (TTX, 10 −7 M) sensitive manner. The reverse Na +/Ca 2+-exchange inhibitor KB-R7943 (3×10 −5 M) failed to inhibit the stimulation-evoked release of [ 3 H ]NA, but increased the resting outflow of neurotransmitter. The ‘N-type’ voltage-sensitive Ca 2+-channel (VSCC) blocker ω-conotoxin (ω-CgTx) GVIA (10 −8 M) significantly and irreversibly inhibited the release of [ 3 H ]NA on stimulation (∼60–70%). The ‘residual release’ of NA was abolished either by TTX or by reducing external Ca 2+ from 2.5 to 0.25 mM. The ‘residual release’ of NA was also blocked by the non-selective VSCC-blocker neomycin (3×10 −3 M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic α 2-receptor blocker yohimbine (3×10 −7 M). When the release of [ 3 H ]NA was blocked by ω-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na +-pump by removal of K + from the external medium increased both the resting and the axonal stimulation-evoked release of [ 3 H ]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after ω-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca 2+-removal (+1 mM EGTA). Similarly, external Li + (113 mM) or the reverse Na +/Ca 2+ exchange blocker KB-R7943 (3×10 −5 M) significantly inhibited the stimulation-induced transmitter release in ‘K +-free’ solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na +-pump was inhibited in the absence of functioning VSCCs, yohimbine (3×10 −7 M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10 −6 M), an agonist of presynaptic α 2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li +-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na +/Ca 2+-exchange and release neurotransmitter in Na +-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na +/Ca 2+-exchange may also be regulated by presynaptic α 2-receptors.

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings

It was previously reported that the K + -evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or d -serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or d -serine on the K + -evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K + -depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca 2+ -free medium and with voltage-sensitive Ca 2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30–50%) dependent on external Ca 2+ and Ca 2+ channel activation. In contrast, the alkalinization-evoked release of [ 3 H ]noradrenaline was highly sensitive to external Ca 2+ removal and to blockade of Ca 2+ channels with ω-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca 2+ -independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.

Tricyclic antidepressant imipramine reduces the insulin secretory rate in islet cells of Wistar albino rats through a calcium antagonistic action.

Treatments with antidepressants have been associated with modifications in glucose homeostasis. The aim of this study was to assess the effect of imipramine, a tricyclic antidepressant, on insulin-secreting cells. Insulin radioimmunoassay, radioisotopic, fluorimetric and patch-clamp methods were used to characterise the effects of imipramine on ionic and secretory events in pancreatic islet cells from Wistar albino rats. Imipramine induced a dose-dependent decrease in glucose-stimulated insulin output (IC(50)=5.2 micromol/l). It also provoked a concentration-dependent reduction in (45)Ca outflow from islets perifused in the presence of 16.7 mmol/l glucose. Moreover, imipramine inhibited the increase in (45)Ca outflow mediated by K(+) depolarisation. Patch-clamp recordings further revealed that imipramine provoked a marked and reversible decrease of the inward Ca(2+) current. In single islet cells, imipramine counteracted the rise in [Ca(2+)](i) evoked by glucose or high K(+) concentrations. These data indicate that imipramine dose-dependently reduces the insulin secretory rate from rat pancreatic beta cells. Such an effect appears to be mediated by the inhibition of voltage-sensitive Ca(2+) channels with subsequent reduction in Ca(2+) entry. Thus, it is possible that some adverse effects of imipramine are related, at least in part, to its capacity to behave as a Ca(2+) entry blocker.

Activity-dependent hepatocyte growth factor expression and its role in organogenesis and cancer growth suppression

Studies by Murphy et al. have shown that neuronal stimulation can activate immediate early genes that code for transcription factors. Recent data suggest that Ca 2+ elevation in both neuronal cytoplasmic and nuclear compartments is responsible for the coupling of synaptic excitation to gene expression. Deisseroth et al. suggest that Ca 2+ influx through L-type voltage-sensitive Ca 2+ channels (VSCCs) activates cytoplasmic Ca 2+ targets such as calmodulin (CaM). The Ca 2+–CaM complex then translocates to the nucleus leading to Ca 2+ and cAMP response element-binding protein (CREB) phosphorylation and gene expression. Reports have shown that L-type VSCCs are found on the vagus nerve. Other studies have suggested that activation of L-type VSCCs leads to a Ca 2+ store-dependent elevation of nuclear [Ca 2+] that triggers gene expression by more direct activation of nuclear Ca 2+/CaM-dependent protein kinase (CaMK). Moreover, nuclear transcription factors such as DREAM are themselves Ca 2+-dependent, further supporting the importance of both nuclear and cytoplasmic Ca 2+ elevation in regulating gene expression. Our simulation studies suggest that intense synaptic stimulation in combination with amplification by release from intracellular Ca 2+ stores can produce elevations in nuclear Ca 2+ concentration and CaMK phosphorylation leading to CREB phosphorylation and gene expression. One of the downstream events would be the production of hepatocyte growth factor (HGF). HGF has trophic, repair, therapeutic or mitotic effect on kidney, pancreas, spleen, liver, lung, heart and spinal cord. These organs and systems' regeneration can be achieved by either upregulation of HGF release from the vagus nerve or upregulation of HGF production within the system (spinal cord). Conversely, inhibition of HGF release from the vagus nerve can inhibit cancer growth. Vagus nerve seems to be the nerve that nature intends to regulate organ growth and regeneration, it is very possible that other than HGF and injurin, other growth factors could be found in the vagus nerve. Electrical depolarization and hyperpolarization of the vagus nerve would be the most natural and effective way to induce organ regeneration and suppress cancer growth, respectively. A similar pathway seems to exist for different organs as HGF has trophic, repair, therapeutic or mitotic effect on different vagally innervated organs.

Decoding of synaptic voltage waveforms by specific classes of recombinant high-threshold Ca(2+) channels.

Studies suggest that the preferential role of L-type voltage-sensitive Ca(2+) channels (VSCCs) in coupling strong synaptic stimulation to transcription is due to their selective activation of local chemical events. However, it is possible that selective activation of the L-type channel by specific voltage waveforms also makes a contribution. To address this issue we have examined the response of specific Ca(2+) channel types to simulated complex voltage waveforms resembling those encountered during synaptic plasticity (gamma and theta firing frequency). L-, P/Q- and N-type VSCCs (alpha1C, alpha1A, alpha1B/beta1B/alpha2delta, respectively) were all similarly activated by brief action potential (AP) waveforms or sustained step depolarization. When complex waveforms containing large excitatory postsynaptic potentials (EPSPs), APs and spike accommodation were applied under voltage clamp we found that the integrated L-type VSCC current was approximately three times larger than that produced by the P/Q- or N-type Ca(2+) channels (gamma frequency 1 s stimulation). For P/Q- or N-type channels the complex waveforms led to a smaller current than that expected from the response to a simple 1 s step depolarization to 0 or +20 mV. EPSPs present in the waveforms favoured the inactivation of P/Q- and N-type channels. In contrast, activation of the L-type channel was dependent on both EPSP- and AP-mediated depolarization. Expression of P/Q-type channels with reduced voltage-dependent inactivation (alpha1A/beta2A/alpha2delta) or the use of hyperpolarized intervals between AP stimuli greatly increased their response to complex voltage stimuli. We propose that in response to complex synaptic voltage waveforms P/Q- and N-type channels can undergo selective voltage-dependent inactivation leading to a Ca(2+) current mediated predominantly by L-type channels.

Palmitate-induced Ca 2+-signaling in pancreatic beta-cells

Free fatty acids (FFA) have been proposed to participate in the regulation of insulin release from pancreatic beta-cells (β-cells). As a rise in cytosolic free Ca 2+ ([Ca 2+] i ) is a key event for the stimulation of insulin secretion, the effects of saturated FFA on [Ca 2+] i were investigated. Palmitate was used as a reference compound and [Ca 2+] i was measured in single fura-2 loaded HIT-T15 and in primary mouse β-cells. Stimulation of single β-cells with palmitate (100 μM) caused either repetitive Ca 2+ transients or a plateau-like rise in [Ca 2+] i . In HIT-T15 and in mouse β-cells, the number of palmitate-responsive cells, and the amplitude of the palmitate-induced Ca 2+-signals were dependent on the extracellular glucose concentration. In Ca 2+-free medium palmitate (100 μM) caused only 1 or 2 Ca 2+ transients indicating mobilization of Ca 2+ from internal stores. Withdrawal of external Ca 2+, the addition of voltage-sensitive Ca 2+ channel (VSCC) blockers, as well as the K ATP-channel opener diazoxide (100 μM) reversibly blocked the palmitate-induced cytosolic Ca 2+ responses. This demonstrates that Ca 2+ influx through VSCC of the L-type coupled to membrane depolarization through closure of K ATP-channels are crucial for a sustained Ca 2+-signal in response to palmitate. Methyl palmoxirate (100 μM) and 2-bromopalmitate (100 μM), which both inhibit transport of acyl-CoA into the mitochondria, reversibly blocked the palmitate-induced Ca 2+-signals in HIT-T15 as well as in primary mouse β-cells. By contrast, cerulenin (100 μM), an inhibitor of protein acylation, had no effect on the palmitate-induced changes in [Ca 2+] i , which suggests that mitochondrial palmitate metabolism is required for eliciting the Ca 2+-signals. Simultaneous measurement of [Ca 2+] i and the mitochondrial membrane potential (Δ Ψ) revealed palmitate-induced depolarization of Δ Ψ which demonstrates that palmitate does not enhance mitochondrial ATP production. Therefore mitochondrial signals other than ATP appear to be generated from palmitate metabolism that underly the palmitate-induced Ca 2+-signals in pancreatic β-cells.