Insensitive Ca2 Research Articles

Characterization of the Calcium Signaling System in the Submandibular Cell Line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.

Characterization of the Calcium Signaling System in the Submandibular Cell Line SMG‐C6

Abstract. Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG‐C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)‐Ca2+ signaling system in this cell line. Inositol 1,4,5‐trisphosphate(1,4,5‐IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, α1‐adrenergic, P2Y‐nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin‐6 (IL‐6), and tumor necrosis factor‐α (TNFα). Consistently, 1,4,5‐IP3 formation was dramatically stimulated by ACh and ATP. ACh‐stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+‐induced Ca2+ release mechanism is involved in the ACh‐elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3‐sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3‐insensitive stores exist in SMG‐C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store‐operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG‐C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y‐nucleotide receptor agonists through formation of IP3; (ii) both the IP3‐sensitive and ‐insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store‐operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG‐C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells.

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.

Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells.

1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved.

P-558 - The contribution of nifedipine- insensitive Ca2+ influx to Endothelin-1-induced constriction in rat small pulmonary arteries

P-558 - The contribution of nifedipine- insensitive Ca2+ influx to Endothelin-1-induced constriction in rat small pulmonary arteries

Ca2+/calmodulin inhibition and phospholipase C-linked Ca2+ Signaling in clonal beta-cells.

Neurotransmitters and hormones, such as arginine vasopressin (AVP) and bombesin, evoke frequency-modulated repetitive Ca2+ transients in insulin-secreting HIT-T15 cells by binding to receptors linked to phospholipase C (PLC). The role of calmodulin (CaM)-dependent mechanisms in the generation of PLC-linked Ca2+ transients was investigated by use of the naphthalenesulfonamide CaM antagonists W-7 and W-13 and their dechlorinated control analogs W-5 and W-12. W-7 (10-30 microM) and W-13 (30-100 microM), but not W-5 (100 microM) and W-12 (300 microM), reversibly inhibited the AVP- and bombesin-induced Ca2+ transients. As the generation of PLC-linked Ca2+ transients requires mobilization of internal Ca2+ and Ca2+ influx through voltage-sensitive (VSCC) and -insensitive (VICC) Ca2+ channels, the effects of the W compounds on these processes were further investigated. First, W-7 dose dependently diminished K+ (45 mM)-induced Ca2+ signals (IC50, approximately 25 microM), and W-13 (100 microM) reduced the K+ (45 mM)-induced [Ca2+]i rise by about 40-60%, whereas W-5 (100 microM) and W-12 (300 microM) had no effect. In addition, W-7 (100 microM) inhibited whole cell Ca2+ currents in mouse beta-cells by about 60%. Second, pretreatment of cells (5 min) with W-7 (30 microM), but not W-5 (30 microM), inhibited agonist-induced internal Ca2+ mobilization by about 75% in Ca2+-free medium. Neither W-7 (30 microM) nor W-5 (30 microM) affected AVP (100 nM)-stimulated formation of IP3. Third, capacitative Ca2+ influx through VICC activated by thapsigargin (2 microM) in the presence of verapamil (50 microM) was inhibited by W-7 (30 microM) but not by W-5 (30 microM). As all of the W compound effects corresponded well to their reported anticalmodulin activity, a specific anticalmodulin action can be assumed. Thus, Ca2+ via activation of CaM-dependent processes could provide positive feedback on the generation of PLC-linked Ca2+ transients in HIT-T15 cells. This appears to involve CaM-dependent regulation of both mobilization of internal Ca2+ and Ca2+ influx through VSCC and VICC.

5-Hydroxytryptamine induces transient Ca 2+ influx through Ni 2+-insensitive Ca 2+ channels in rat vascular smooth muscle cells

The effects of Ni 2+, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca 2+ dynamics in rat aortic smooth muscle cells were investigated. Ni 2+ (1 mM) significantly inhibited the transient increase in intracellular Ca 2+ concentration ([Ca 2+] i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni 2+ did not suppress the transient increase in Ca 2+ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca 2+ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca 2+ store was almost completely suppressed by Ni 2+. Ni 2+ had no effect on 5-HT-induced inositol triphosphate production and Ca 2+ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca 2+ influx through Ni 2+-insensitive Ca 2+ channels, which are distinguishable from the voltage-dependent or store-operated Ca 2+ channels.

Role of Ca2+-ATPase in spontaneous oscillations of cytosolic free Ca2+in GH3rat pituitary cells

The relative contribution of voltage-sensitive Ca2+channels, Ca2+-ATPases, and Ca2+release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca2+-ATPase expressed in these cells. We show that spontaneous [Ca2+]ioscillations in GH3cells were unaffected by Ca2+depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]ioscillations were highly temperature dependent. Decreasing the temperature from 22 to 17°C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]ioscillations. Furthermore, the rate of ATP-dependent45Ca2+uptake into GH3-derived microsomes was greatly reduced at 17°C. The effect of decreased temperatures on extracellular Ca2+influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+channels, was relatively unchanged at 17°C. These results suggest that in GH3secretory cells, Ca2+influx via L-type Ca2+channels initiates spontaneous [Ca2+]ioscillations, which are then maintained by the combined activity of Ca2+-ATPase and Ca2+-induced Ca2+release from Tg/IP3-insensitive intracellular stores.

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes.

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 +/- 12% (p < 0.05) and 53 +/- 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 +/- 12%) but not AVP (9 +/- 11%). Neither AVP nor ET-1 significantly affected cell number or [3H]thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 +/- 12 nmol/L from a basal value of 77 +/- 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, both stimulates protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.

Bradykinin-evoked Ca 2+ mobilization in Madin Darby canine kidney cells

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca 2+] i) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca 2+] i transient in a dose-dependent manner, measured by fura-2 fluorimetry and digital video imaging. The transient consisted of a rise and a decay and [Ca 2+] i returned to baseline without oscillations. External Ca 2+ influx occurred, as demonstrated by Mn 2+ quench and external Ca 2+ removal measurements. Bradykinin acted by stimulating bradykinin B 2 receptors as evidenced by blockade by d-arginyl- l-arginlyl- l-prolyl- trans-4-hydroxy- l-prolylglycyl-3-(2-thienyl)- l-alanyl- l-seryl- d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl- l-(2α,3β,7aβ)-octahydro-1 H-indole-2-carbonyl- l-arginine (HOE 140) but not by d-arginyl- l-arginlyl- l-prolyl- trans-4-hydroxy- l-prolylglycyl-3-(2-thienyl)- l-alanyl- l-seryl- d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl- l-(2α,3β,7aβ)-octahydro-1 H-indole-2-carbonyl ([Des–Arg]HOE 140). The [Ca 2+] i signal was abolished by 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca 2+ from internal stores since it was abolished by pretreatment with thapsigargin or cyclopiazonic acid. The mobilization of the internal Ca 2+ store subsequently triggered a 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride (SKF 96365)-insensitive Ca 2+ entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-β-naphthylamide did not alter the transient, thus excluding the participation of mitochondria and lysosomes. Efflux via Ca 2+ pumps contributed to the decay of the transient. Efflux via Na +/Ca 2+ exchange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 12-myristate 13-acetate, and was enhanced by the protein kinase C inhibitors sphingosine and chelerythrine, the protein kinase A inhibitor 2,5-di-( t-butyl)-1,4-hydroquinone, N-[2-( p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and agents that elevated levels of 3′,5′-cyclic guanosine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP.

Long-term induction of a unique C1- current by endothelin-1 in an epithelial cell line from rat lung: evidence for regulation of cytoplasmic calcium.

1. Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura-2, we investigated the relationship between the cell membrane potential, whole-cell currents and the free cytoplasmic Ca2+ concentration ([Ca2+]i) in response to 10 nM endothelin-1 (ET) in a rat respiratory epithelial cell line (L2). 2. Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+ replacement with N-methyl-D-glucamine+ (NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low C1- (6 mM), resulting in a complete breakdown of Vm. 3. In perforated patch clamp experiments, the ET-induced whole-cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of -22.7 mV. IET was reduced by 85 % in low C1- (6 mM), but was unaffected by Ca2+ removal, Na+ replacement with NMDG+, pipette K+ replacement with Cs+ or 1 mM Ni2+ in the bath. 4. IET was unaffected by (+)-isradipine (100 nM), a specific L-type Ca2+ channel (L-VDCC) blocker. Transient inward Sr2+ currents through L-VDCCs were blocked by ET. 5. ET induced a biphasic Ca2+ signal, consisting of a 'peak' and a 'plateau' elevation of [Ca2+]i. Simultaneous patch clamp and fura-2 measurements revealed that IET coincided with intracellular Ca2+ release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]i was prevented by pretreatment with thapsigargin in a Ca2+-free bath, both activation time and amplitude of IET were reduced. Under these conditions, ET caused a decrease of [Ca2+]i. 6. The C1- channel blocker mefenamic acid (MFA) had a dual, concentration-dependent effect on both IET and the ET-induced 'plateau' elevation of [Ca2+]i: an increase at 10 microM, but an almost complete block at 100 microM. The effect of MFA on IET preceded the effect on [Ca2+]i. 7. The ET-induced 'plateau' [Ca2+]i fell below control values in a low-C1- (6 mM) solution. 8. These data indicate an amplifying function of intracellular Ca2+ release on an otherwise Ca2+-independent, unique C1- current by ET. Moreover, this C1- current appears to be functionally coupled with dihydropyridine (DHP)-insensitive Ca2+ entry, suggesting a modulatory role for long-lasting effects of ET.

Alterations in Ca 2+ storage and mobilization in submandibular acinar cells of reserpine-treated rats

The present study investigated the effects of the catecholamine-depleting drug reserpine on cellular Ca 2+ storage and mobilization in rat submandibular acinar cells. Adult rats received seven daily injections of reserpine (0.5 mg/kg) and inositol 1,4,5-trisphosphate (IP 3) formation and Ca 2+ mobilization were measured in isolated submandibular acinar cells. Ultrastructural analysis demonstrated a significant reduction in rough endoplasmic reticulum (ER) and a dramatic accumulation of secretory granules in the cells of treated animals. Reserpine reduced acetylcholine (ACh)-stimulated IP 3 formation by 46% and the initial increase in cytosolic Ca 2+ concentration ([Ca 2+] i) in response to ACh or thapsigargin was reduced by 21 and 56%, respectively. While norepinephrine (NE) did not induce significant IP 3 formation, the [Ca 2+] i response to NE was increased 360% by reserpine treatment. Reserpine treatment also enhanced the sustained [Ca 2+] i increase following these stimuli. After stimulation with ACh or NE, exposure to ionomycin caused a further elevation in [Ca 2+] i which was significantly larger in the cells of treated animals. After exposure to agonist+ionomycin, addition of monensin induced a third increase in [Ca 2+] i which was significantly larger in cells of reserpine-treated animals. While capacitative Ca 2+ entry was not altered, NE-activated Ca 2+ influx was abolished after reserpine treatment. Reserpine treatment therefore alters IP 3-sensitive and insensitive Ca 2+ stores, non-capacitative Ca 2+ influx and active Ca 2+ transport in submandibular acinar cells of rats.

Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line

The Drosophila melanogaster genes, transient receptor potential ( trp) and transient receptor potential-like ( trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca 2+ levels measured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system. Activation of DM1 receptor in S2-DM1-TRPL cells by 100 μM carbamylcholine induced Ca 2+ release from an intracellular Ca 2+ pool followed by a Gd 3+-insensitive Ca 2+ influx. Pretreatment of S2-DM 1-TRPL cells with 10 μM atropine abolished Gd 3+-insensitive Ca 2+ influx triggered by carbamylcholine, but the response was not blocked by prior incubation with pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 μM thapsigargin for 10 min or 100 nM thapsigargin for 60 min in Ca 2+-free solution. In some cells, TRPL channels activated by thapsigargin could further be activated by carbamylcholine. The findings suggest that, when stably expressed in the S2 cell line, TRPL may be regulated by two distinct mechanisms: (i) store depletion; and (ii) stimulation of DM1 receptor via pertussis-toxin insensitive G-protein (or the subsequent activation of PLC), but without further requirement for Ca 2+ release.

M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons.

Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (</=300 nM) and m4-toxin (</=100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of IBa by m4-toxin gave an IC50 of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage-activated Ca2+ channels in rat intracardiac neurons is mediated by the M4 muscarinic receptor. M4 receptor activation shifted the voltage dependence and depressed maximal activation of Ca2+ channels but had no effect on the steady-state inactivation of Ca2+ channels. Peak Ca2+ channel tail current amplitude was reduced >/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.

Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells.

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.

Evidence for a Na +Ca 2+ exchanger in rat pancreatic ducts

Only recently has it been recognized that intracellular Ca 2+ is an important cellular mediator in pancreatic ducts. The aim of the present study was to characterize the Call efflux pathway in ducts freshly prepared from rat pancreas. Lowering of extracellular Na + concentration resulted in a significant increase in intracellular Ca 2+. This effect was fast, reversible, dependent on the extracellular Na+ concentration and did not correlate with intracellular pH changes. It was abolished in Ca 2+-free solutions, indicating that the outwardly directed Na + gradient was directly coupled to a flufenamate insensitive Ca 2+ influx. Removal and reintroduction of extracellular Na + induced transient hyperpolarization and depolarization of V m, respectively. Taken together, our data indicate that pancreatic ducts possess an electrogenic Na +Ca 2+ exchanger, which under control conditions is responsible for transporting Ca 2+ out of resting duct cells.

SR Ca2+ pump heterogeneity in coronary artery: free radicals and IP3-sensitive and -insensitive pools.

Reactive oxygen species are known to decrease the action of agents that mobilize Ca2+ from sarcoplasmic reticulum (SR) in pig coronary artery smooth muscle. Potentially, this may be due to damage to the SR Ca2+ pump or to the myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release channels. Here we report on the effects of peroxide and superoxide on the SR Ca2+ pump and the subsequent IP3-induced Ca2+ release. Smooth muscle cells cultured from pig left coronary arteries were permeabilized using saponin and then loaded with 45Ca2+ in the presence of an ATP-regenerating system and the mitochondrial Ca2+ uptake inhibitor sodium azide. IP3 caused a release of up to 65% of the loaded 45Ca2+, whereas the Ca2+ ionophore A-23187 caused a release of > 95%. The nature of the IP3-insensitive component of the Ca2+ uptake is not known. The IP3-induced Ca2+ release occurred at 0 or 37 degrees C and was complete in < 30 s. The 50% effective concentration for IP3 was 2.7 +/- 1.0 microM at pH 6.8 and 37 degrees C. At pH 7.4 the IP3-induced Ca2+ release was slightly lower than at pH 6.4-6.8. The IP3-induced release was also inhibited by Ca2+ concentration in the release medium. To investigate the effects of peroxide or superoxide, the cells were treated with these agents, washed, skinned, and then used to examine the IP3-sensitive and -insensitive Ca2+ pools under the conditions in which the IP3-sensitive pool was 60-65% of the total. Peroxide pretreatment was equipotent in inhibiting loading into the IP3-sensitive and -insensitive Ca2+ pools. In contrast, superoxide pretreatment inhibited loading into the IP3-sensitive pool but not into the IP3-insensitive pool. These data are consistent with a model in which the SR Ca2+ pumps are heterogeneous: those required to pump Ca2+ into the IP3-sensitive pool are inhibited by peroxide and superoxide, but those loading the IP3-insensitive pool are inhibited by peroxide only.

TXA2 agonists inhibit high-voltage-activated calcium channels in rat hippocampal CA1 neurons.

Whole cell voltage clamp recordings were used to investigate the effects of thromboxane A2 (TXA2) agonists on the voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. TXA2 agonists [1S-[1 alpha, 2 beta(5Z), 3 alpha(1E, 3S*)4 alpha ]]-7-[3-[3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo [2,2,1]heptan-2-yl]-5-heptenoic acid (I-BOP) and U-46619, reversibly suppressed the whole cell Ca2+ currents in a concentration-dependent manner. The effect was blocked by specific TXA2 receptor antagonist, SQ-29548. I-BOP as well as U-46619 inhibited both omega-conotoxin GVIA (CgTx)-sensitive and nimodipine sensitive Ca2+ currents but had no effect on CgTx/nimodipine insensitive Ca2+ currents. The I-BOP and U-46619 inhibition of Ca2+ currents was blocked by internal dialysis of hippocampal neurons with specific protein kinase C (PKC) inhibitors, NPC-15437 and PKC inhibitor-(19-36). Pretreatment of hippocampal neurons with either 5 micrograms/ml pertussis toxin (PTX) or 5 micrograms/ml cholera toxin (CTX) did not significantly affect the suppression of the Ca2+ currents by I-BOP and U-46619. Dialyzing with 1 mM guanosine 5'-O-(3-thiotriphosphate) or 1 mM GDP significantly attenuated the I-BOP or U-46619 action. These results demonstrate that TXA2 agonists inhibit both CgTx- and nimodipine-sensitive Ca2+ currents but not CgTx/nimodipine-insensitive currents in rat hippocampal CA1 neurons via a PTX- and CTX-insensitive G protein-coupled activation of the PKC pathway.

Molecular Determinants of High Affinity Dihydropyridine Binding in L-type Calcium Channels

The pore-forming alpha1 subunit of L-type voltage-gated Ca2+ channels is pharmacologically modulated by dihydropyridine (DHP) Ca2+ antagonists and agonists. Site-directed mutation of amino acids within transmembrane segments IIIS6 and IVS6 to those characteristic of DHP-insensitive channels revealed 2 mutations in IIIS6 (I1049F and I1052F) and 4 mutations in IVS6 (Y1365I, M1366F, I1372M, and I1373L) with increased KD values for (+)-[3H]PN200-110 binding. A tyrosine residue (Y1048) in IIIS6 that is conserved between DHP-sensitive and -insensitive Ca2+ channels was also altered by mutagenesis. Y1048F had a KD for (+)-[3H]PN200-110 binding that was increased 12-fold, and Y1048A had a KD at least 1000-fold higher than that of wild-type. These results support the hypothesis that transmembrane segments IIIS6 and IVS6 both contribute critical amino acid residues to the DHP receptor site and that Tyr-1048 within transmembrane segment IIIS6 is required for high affinity DHP binding, even though it is conserved between DHP-sensitive and -insensitive Ca2+ channels.

Properties of the sarcoplasmic reticulum Ca(2+)-pump in coronary artery skinned smooth muscle.

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca(2+)-uptake which increased linearly with time for > 1 h. The Ca(2+)-uptake occurred with Km values of 0.20 +/- 0.03 microM for Ca2+ and 400 +/- 34 microM for MgATP2-. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC50 values of 0.13 +/- 0.02 and 0.56 +/- 0.04 microM, respectively. These properties of SR Ca(2+)-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4-6.8).