The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

Calcium (Ca(2+)) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca(2+) permeable channels, Ca(2+) pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca(2+) levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca(2+) signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca(2+) channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca(2+) release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca(2+) signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca(2+) channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca(2+) release channels, pumps and Ca(2+)-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2a pump and reciprocally regulate isoforms of the ca(2+)/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca(2+) signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca(2+) signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.     

GABA(B) receptor-mediated ERK1/2 phosphorylation via a direct interaction with Ca(V)1.3 channels

Neuronal L-type Ca(2+) channels play pivotal roles in regulating gene expression, cell survival, and synaptic plasticity. The Ca(V)1.2 and Ca(V)1.3 channels are 2 main subtypes of neuronal L-type Ca(2+) channels. However, the specific roles of Ca(V)1.2 and Ca(V)1.3 in L-type Ca(2+) channel-mediated neuronal responses and their cellular mechanisms are poorly elucidated. On the basis of our previous study demonstrating a physical interaction between the Ca(V)1.3 channel and GABA(B) receptor (GABA(B)R), we further examined the involvement of Ca(V)1.2 and Ca(V)1.3 in the GABA(B)R-mediated activation of ERK(1/2), a kinase involved in both CREB activation and synaptic plasticity. After confirming the involvement of L-type Ca(2+) channels in baclofen-induced ERK(1/2) phosphorylation, we examined a specific role of Ca(V)1.2 and Ca(V)1.3 channels in the baclofen effect. Using siRNA-mediated silencing of Ca(V)1.2 or Ca(V)1.3 messenger, we determined the relevance of each channel subtype to baclofen-induced ERK(1/2) phosphorylation in a mouse hippocampal cell line (HT-22) and primary cultured rat neurons. In the detailed characterization of each subtype using HEK293 cells transfected with Ca(V)1.2 or Ca(V)1.3, we found that GABA(B)R can increase ERK(1/2) phosphorylation and Ca(V)1.3 channel activity through direct interaction with Ca(V)1.3 channels. These results suggest a functional interaction between Ca(V)1.3 and GABA(B)R and important implications of Ca(V)1.3/GABA(B)R clusters for translating synaptic activity into gene expression alterations.     

T-type Ca2+ channels mediate propagation of odor-induced Ca2+ transients in rat olfactory receptor neurons

Propagation of odor-induced Ca(2+) transients from the cilia/knob to the soma in mammalian olfactory receptor neurons (ORNs) is thought to be mediated exclusively by high-voltage-activated Ca(2+) channels. However, using confocal Ca(2+) imaging and immunocytochemistry we identified functional T-type Ca(2+) channels in rat ORNs. Here we show that T-type Ca(2+) channels in ORNs also mediate propagation of odor-induced Ca(2+) transients from the knob to the soma. In the presence of the selective inhibitor of T-type Ca(2+) channels mibefradil (10-15 microM) or Ni(2+) (100 microM), odor- and forskolin/3-isobutyl-1-methyl-xanthine (IBMX)-induced Ca(2+) transients in the soma and dendrite were either strongly inhibited or abolished. The percentage of inhibition of the Ca(2+) transients in the knob, however, was 40-50% less than that in the soma. Ca(2+) transients induced by 30 mM K(+) were partially inhibited by mibefradil, but without a significant difference in the extent of inhibition between the knob and soma. Furthermore, an increase of as little as 2.5 mM in the extracellular K(+) concentration (7.5 mM K(+)) was found to induce Ca(2+) transients in ORNs, and such responses were completely inhibited by mibefradil or Ni(2+). Total replacement of extracellular Na(+) with N-methyl-d-glutamate inhibited none of the odor-, forskolin/IBMX- or 7.5 mM K(+)-induced Ca(2+) transients. Positive immunoreactivity to the Ca(v)3.1, Ca(v)3.2 and Ca(v)3.3 subunits of the T-type Ca(2+) channel was observed throughout the soma, dendrite and knob. These data suggest that involvement of T-type Ca(2+) channels in the propagation of odor-induced Ca(2+) transients in ORNs may contribute to signal transduction and odor sensitivity.     

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.     

Differential effects of a green tea-derived polyphenol (−)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle

(-)-Epigallocatechin-3-gallate (EGCg), a green tea-derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined its effects on the acidosis-induced change in the Ca(2+) sensitivity of myofilaments in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. EGCg (0.1 mM) significantly inhibited the decrease caused by lowering the pH from 7.0 to 6.0 in the Ca(2+) sensitivity of myofibrillar ATPase activity in cardiac muscle, but not in skeletal muscle. Studies on recombinant mouse cardiac troponin C (cTnC) and chicken fast skeletal troponin C (sTnC) using circular dichroism and intrinsic and extrinsic fluorescence spectroscopy showed that EGCg bound to cTnC with a dissociation constant of approximately 3-4 muM, but did not bind to sTnC. By presumably binding to the cTnC C-lobe, EGCg decreased Ca(2+) binding to cTnC and overcame the depressant effect of protons on the Ca(2+) sensitivity of the cardiac contractile response. To demonstrate isoform-specific effects of the action of EGCg, the pH sensitivity of the Ca(2+) response was examined in cardiac myofibrils in which endogenous cTnC was replaced with exogenous sTnC or cTnC and in skeletal myofibrils in which the endogenous sTn complex was replaced with whole cardiac Tn complex (cTn). The results suggest that the binding of EGCg to the cardiac isoform-specific TnC or Tn complex alters the effect of pH on myofilament Ca(2+) sensitivity in striated muscle.     

Inhibition effect of arachidonic acid on hypoxia-induced [Ca(2+)](i) elevation in PC12 cells and human pulmonary artery smooth muscle cells

[Ca(2+)](i) elevation is a key event when O(2) sensitive cells, e.g. PC12 cells and pulmonary artery smooth muscle cells, face hypoxia. Ca(2+) entry pathways in mediating hypoxia-induced [Ca(2+)](i) elevation include: voltage-gated Ca(2+) channels (VGCCs), transient receptor potential (TRP) channel and Na(+)-Ca(2+) ex-changer (NCX). In the pulmonary artery, accumulated evidence strongly suggests that prostaglandins (PGs) are involved in pulmonary inflammation and cause vasoconstriction during hypoxia. In this study, we investigated the effect of arachidonic acid (AA), the upstream substrate for PGs, on hypoxia response in O(2) sensitive cells. Exogenous application of AA significantly inhibited hypoxia-induced [Ca(2+)](i) elevation. This effect was due to AA itself rather than its degenerative products. The pharmacological modulation of endogenous AA showed that the prevention of AA generation by blockage of cPLA2, diacylglycerol (DAG) lipase and fatty acid hydrolysis (FAAH), augments hypoxia-induced [Ca(2+)](i) elevation, whereas prevention of AA degeneration attenuates hypoxia-induced [Ca(2+)](i) elevation. Over-expression of COX2 enhances hypoxia-induced [Ca(2+)](i) elevation and this enhancement is reversed by exogenous AA. Our results suggest that AA inhibits hypoxia response. The dynamic alterations in cellular lipids might determine cell response to hypoxia.     

Effects of increasing Ca2+ channel-vesicle separation on facilitation at the crayfish inhibitory neuromuscular junction

We investigated the mechanism of facilitation at the crayfish inhibitory neuromuscular junction before and after blocking P-type Ca(2+) channels. P-type channels have been shown to be closer to releasable synaptic vesicles than non-P-type channels at this synapse. Prior to the block of P-type channels, facilitation evoked by a train of 10 action potentials at 100 Hz was increased by application of 40 mM [Mg(2+)](o), but decreased by pressure-injected EGTA. Blocking P-type channels with 5 nM omega-Aga IVA, which reduced total Ca(2+) influx and release to levels comparable to that recorded in 40 mM [Mg(2+)](o), did not change the magnitude of facilitation. We explored whether this observation could be attributed to the buffer saturation model of facilitation, since increasing the Ca(2+) channel-vesicle separation could potentially enhance the role of endogenous buffers. The characteristics of facilitation in synapses treated with omega-Aga IVA were probed with broad action potentials in the presence of K(+) channel blockers. After Ca(2+) channel-vesicle separation was increased by omega-Aga IVA, facilitation probed with broad action potential was still decreased by EGTA injection and increased by 40 mM [Mg(2+)](o). EGTA-AM perfusion was used to test the impact of EGTA over a range of concentration in omega-Aga IVA-poisoned preparations. The results showed a concentration dependent decrease in facilitation as EGTA concentration rose. Thus, probing facilitation with EGTA and reduced Ca(2+) influx showed that characteristics of facilitation are not changed after the role of endogenous buffer is enhanced by increasing Ca(2+) channel-vesicle separation. There is no clear indication that buffer saturation has become the dominant mechanism for facilitation after omega-Aga IVA poisoning. Finally, we sought correlation between residual Ca(2+) and the magnitude of facilitation. Using fluorescence transients of a low affinity Ca(2+) indicator, we calculated the ratio of fluorescence amplitude measured immediately before test pulse (residual Ca(2+)) to that evoked during action potential (local Ca(2+)). This ratio provides an estimate of relative changes between residual Ca(2+) and local Ca(2+) important for release. There is a significant increase in the ratio when Ca(2+) influx is reduced by 40 mM [Mg(2+)](o). The magnitude of facilitation exhibited a clear and positive correlation with the ratio, regardless of separation between Ca(2+) channels and releasable vesicles. This correlation suggests the importance of relative changes between residual and local Ca(2+) and lends support to the residual Ca(2+) hypothesis of facilitation.     

Electrophysiological and immunofluorescence characterization of Ca(2+) channels of acutely isolated rat sphenopalatine ganglion neurons

The sphenopalatine ganglion (SPG) is the main parasympathetic ganglion that is involved in regulating cerebral vascular tone and gland secretion. SPG neurons have been implicated in some types of migraine headaches but their precise role has yet to be determined. In addition, very little information is available regarding ion channel modulation by neurotransmitters that are involved in the parasympathetic drive of SPG neurons. In this study, acute isolation of adult rat SPG neurons was developed in order to begin the electrophysiological characterization of this ganglion. Under our dissociation conditions, the average number of neurons obtained per ganglion was greater than 1200. Immunofluorescence imaging results showed positive labeling with acetylcholinesterase (AChE), confirming the parasympathetic nature of SPG neurons. On the other hand, weak tyrosine hydroxylase immunostaining was observed in these neurons. Whole-cell patch-clamp recordings revealed that most of the Ca(2+) current is carried by N-type (53%) and SNX-482 resistant R-type (30%) Ca(2+) channels. In addition, Ca(2+) currents were inhibited in a voltage-dependent manner following exposure to oxotremorine-M (Oxo-M), norepinephrine and ATP via muscarinic acetylcholine receptor 2 (M(2) AChR) subtype, adrenergic and P2Y purinergic receptors, respectively. The peptides VIP and angiotensin II failed to modulate Ca(2+) currents, suggesting that these receptors are not present on the SPG soma or do not couple to Ca(2+) channels. In summary, our data suggest that the Ca(2+) current inhibition mediated by Oxo-M, NE and ATP in adult rat SPG neurons plays an integral part in maintaining parasympathetic control of cranial functions.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

Role of TRP channels and NCX in mediating hypoxia-induced [Ca(2+)](i) elevation in PC12 cells

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.     

Cellular mechanisms underlying the effect of a single exposure to neonatal handling on neurotrophin-3 in the brain of 1-day-old rats

Neurotrophin-3 (NT-3) has an important role in brain development and is thus a good candidate molecule to be involved in the cellular mechanisms mediating the effects of early experiences on the brain. In the present work we employed the model of neonatal handling, which is known to affect the ability of the adult organism to respond to stressful stimuli, and determined its effects on NT-3 levels in the rat hippocampus and cortex 2, 4 and 8 h after handling on postnatal day 1. We also recorded maternal behavior during the 8 h following handling. At both the 4 and 8 h time-points there was an increase in NT-3 positive cells in field 1 of Ammon's horn (CA1 area of the hippocampus) and parietal cortex of the handled animals. In the parietal cortex NT-3 levels increased with time following handling: at 8 h there were more NT-3 positive cells than at 4 h. During the 4 h following the end of handling, handled pups were subject to more maternal licking, indicating that the more intense maternal care could underlie the handling-induced increase in NT-3. In the hippocampus, the handling induced increase in NT-3 was cancelled by inhibition of N-methyl-D-aspartate (NMDA), AMPA/kainate, or GABA-A receptors, as well as L-type voltage-gated Ca(2+) channels. It thus appears that neonatal handling activates these neurotransmitter receptors and channels, leading to increased intracellular Ca(2+) and increased NT-3 expression. NT-3 can then activate downstream effectors and exert its morphogenetic actions and thus imprint the effects of handling on the brain.     

Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice

The aim of the present study was to establish whether alterations in sarcoplasmic reticulum function are involved in the abnormal Ca(2+) homeostasis of skeletal muscle in mice with muscular dystrophy ( mdx). The properties of the sarcoplasmic reticulum and contractile proteins of fast- and slow-twitch muscles were therefore investigated in chemically skinned fibres isolated from the extensor digitorum longus (EDL) and soleus muscles of normal (C57BL/10) and mdx mice at 4 and 11 weeks of development. Sarcoplasmic reticulum Ca(2+) uptake, estimated by the Ca(2+) release following exposure to caffeine, was significantly slower in mdx mice, while the maximal Ca(2+) quantity did not differ in either type of skeletal muscle at either stage of development. In 4-week-old mice spontaneous sarcoplasmic reticulum Ca(2+) leakage was observed in EDL and soleus fibres and this was more pronounced in mdx mice. In addition, the maximal Ca(2+)-activated tension was smaller in mdx than in normal fibres, while the Ca(2+) sensitivity of the contractile apparatus was not significantly different. These results indicate that mdx hindlimb muscles are affected differently by the disease process and suggest that a reduced ability of the Ca(2+)-ATPase to load Ca(2+) and a leaky sarcoplasmic reticulum membrane may be involved in the altered intracellular Ca(2+) homeostasis.     

Sub-cellular Ca2+ dynamics affected by voltage- and Ca2+-gated K+ channels: Regulation of the soma-growth cone disparity and the quiescent state in Drosophila neurons

Using Drosophila mutants and pharmacological blockers, we provide the first evidence that distinct types of K(+) channels differentially influence sub-cellular Ca(2+) regulation and growth cone morphology during neuronal development. Fura-2-based imaging revealed in cultured embryonic neurons that the loss of either voltage-gated, inactivating Shaker channels or Ca(2+)-gated Slowpoke BK channels led to robust spontaneous Ca(2+) transients that preferentially occurred within the growth cone. In contrast, loss of voltage-gated, non-inactivating Shab channels did not show such a disparity and sometimes produced soma-specific Ca(2+) transients. The fast spontaneous transients in both the soma and growth cone were suppressed by the Na(+) channel blocker tetrodotoxin, indicating that these Ca(2+) fluctuations stemmed from increases in membrane excitability. Similar differences in regional Ca(2+) regulation were observed upon membrane depolarization by high K(+)-containing saline. In particular, Shaker and slowpoke mutations enhanced the size and dynamics of the depolarization-induced Ca(2+) increase in the growth cone. In contrast, Shab mutations greatly prolonged the Ca(2+) increase in the soma. Differential effects of these excitability mutations on neuronal development were indicated by their distinct alterations in growth cone morphology. Loss of Shaker currents increased the size of lamellipodia and the number of filopodia, structures associated with the actin cytoskeleton. Interestingly, loss of Slowpoke currents strongly influenced tubulin regulation, enhancing the number of microtubule loop structures per growth cone. Together, our findings support the idea that individual K(+) channel subunits differentially regulate spontaneous sub-cellular Ca(2+) fluctuations in growing neurons that may influence activity-dependent growth cone formation.     

Functional TRPV4 channels are expressed in mouse skeletal muscle and can modulate resting Ca2+ influx and muscle fatigue

Skeletal muscle contraction is basically controlled by Ca(2+) release and its reuptake into the sarcoplasmic reticulum. However, the long-term maintenance of muscle function requires an additional Ca(2+) influx from extracellular. Several mechanisms seem to contribute to the latter process, such as store-operated Ca(2+) entry, stretch-activated Ca(2+) influx and resting Ca(2+) influx. Candidate channels that may control Ca(2+) influx into muscle fibers are the STIM proteins, Orai, and the members of the transient receptor potential (TRP) family of cation channels. Here we show that TRPV4, an osmo-sensitive cation channel of the vanilloid subfamily of TRP channels is functionally expressed in mouse skeletal muscle. Western blot analysis showed the presence of TRPV4-specific bands at about 85 and 100?kDa in all tested muscles. The bands were absent when muscle proteins from TRPV4 deficient mice were analyzed. Using the manganese quench technique, we studied the resting influx of divalent cations into isolated wild-type muscle fibers. The specific TRPV4-channel activator 4α-phorbol-12,13-didecanoate (4α-PDD) stimulated resting influx by about 60% only in wild-type fibers. Electrical stimulation of soleus muscles did not reveal changes in isometric twitch contractions upon application of 4α-PDD, but tetanic contractions (at 120?Hz) were slightly increased by about 15%. When soleus muscles were stimulated with a fatigue protocol, muscle fatigue was significantly attenuated in the presence of 4α-PDD. The latter effect was not observed with muscles from TRPV4(-/-) mice. We conclude that TRPV4 is functionally expressed in mouse skeletal muscle and that TRPV4 activation modulates resting Ca(2+) influx and muscle fatigue.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.     

Evidence for two calcium currents in insulin-secreting cells

Inward currents were measured in cultured rat pancreatic islet cells using the whole-cell patch-clamp technique. Depolarization elicited both transient and more sustained inward currents. The transient current was blocked by external Na removal, TTX or depolarizing holding potentials while the sustained current was blocked by external Ca removal or the addition of Co, Ni or Cd ions to the external solution. Replacing Ca with Ba increased the amplitude of the sustained current and decreased its rate of decay. These results suggest that the transient current was due to the activation of Na channels while the sustained current was mediated by Ca channels. Two characteristics of the data suggested that two Ca currents may contribute to the sustained current. First, a second shoulder, or hump, was observed on the descending limb of the Ca current I–V of many cells, suggesting that they may possess two Ca channels that activate at different voltages. Second, the low and high voltage components of the I–V were differentially suppressed by Cd. Neither of the Ca current components was very sensitive to the Ca channel agonist BAY K 8644. These results were confirmed in HIT cells, another insulinsecreting preparation.     

Potential contribution of a voltage-activated proton conductance to acid extrusion from rat hippocampal neurons

We examined the potential contribution of a voltage-gated proton conductance (gH+) to acid extrusion from cultured postnatal rat hippocampal neurons. In neurons loaded with Ca2+- and/or pH-sensitive fluorophores, transient exposures to 25-139.5 mM external K+ (K+o) or 20 microM veratridine in the presence of 2 mM Ca2+o (extracellular pH (pHo) constant at 7.35) caused reversible increases and decreases in intracellular free calcium concentration ([Ca2+]i) and intracellular pH (pHi), respectively. In contrast, under external Ca2+-free conditions, the same stimuli failed to affect [Ca2+]i but caused an increase in pHi, the magnitude of which was related to the [K+]o applied and the change in membrane potential. Consistent with the properties of gH+s in other cell types, the magnitude of the rise in pHi observed in the absence of external Ca2+ was not affected by the removal of external Na+ but was sensitive to external Zn2+ and temperature and was dependent on the measured transmembrane pH gradient (DeltapHmemb). Increasing DeltapH(memb) by pretreatment with carbonylcyanide-p-trifluoromethoxyphenylhydrazone augmented both the high-[K+]o-evoked rise in pHi and the Zn2+-sensitive component of the rise in pHi, suggestive of increased acid extrusion via a gH+. The inhibitory effect of Zn2+ at a given DeltapHmemb was further enhanced by increasing pHo from 7.35-7.8, consistent with a pHo-dependent inhibition of the putative gH+ by Zn2+. Under conditions designed to isolate H+ currents, a voltage-dependent outward current was recorded from whole-cell patch-clamped neurons. Although the outward current appeared to show some selectivity for protons, it was not sensitive to Zn2+ or temperature and the H+-selective component could not be separated from a larger conductance of unknown selectivity. Nonetheless, taken together, the results suggest that a Zn2+-sensitive proton conductive pathway is present in rat hippocampal neurons and contributes to H+ efflux under depolarizing conditions.     

Involvement of Na+-Ca2+ exchanger on metabotropic glutamate receptor 1-mediated [Ca2+]i transients in rat cerebellar Purkinje neurons

Cerebellar Purkinje neurons have intracellular regulatory systems including Ca2+-binding proteins, intracellular Ca2+ stores, Ca2+-ATPase and Na+-Ca2+ exchanger (NCX) that keep intracellular Ca2+ concentration ([Ca2+]i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca2+]i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.