The Ca2+ channel beta3 subunit differentially modulates G-protein sensitivity of alpha1A and alpha1B Ca2+ channels

We have shown previously that the Ca2+ channel beta3 subunit is capable of modulating tonic G-protein inhibition of alpha1A and alpha1B Ca2+ channels expressed in oocytes. Here we determine the modulatory effect of the Ca2+ channel beta3 subunit on M2 muscarinic receptor-activated G-protein inhibition and whether the beta3 subunit modulates the G-protein sensitivity of alpha1A and alpha1B currents equivalently. To compare the relative inhibition by muscarinic activation, we have used successive ACh applications to remove the large tonic inhibition of these channels. We show that the resulting rebound potentiation results entirely from the loss of tonic G-protein inhibition; although the currents are temporarily relieved of tonic inhibition, they are still capable of undergoing inhibition through the muscarinic pathway. Using this rebound protocol, we demonstrate that the inhibition of peak current amplitude produced by M2 receptor activation is similar for alpha1A and alpha1B calcium currents. However, the contribution of the voltage-dependent component of inhibition, characterized by reduced inhibition at very depolarized voltage steps and the relief of inhibition by depolarizing prepulses, was slightly greater for the alpha1B current than for the alpha1A current. After co-expression of the beta3 subunit, the sensitivity to M2 receptor-induced G-protein inhibition was reduced for both alpha1A and alpha1B currents; however, the reduction was significantly greater for alpha1A currents. Additionally, the difference in the voltage dependence of inhibition of alpha1A and alpha1B currents was heightened after co-expression of the Ca2+ channel beta3 subunit. Such differential modulation of sensitivity to G-protein modulation may be important for fine tuning release in neurons that contain both of these Ca2+ channels.     

cAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type alpha 1 subunit as evidenced by a 40-60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type alpha 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.     

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function

Literature review for the years 1997 and 1998 presents new concepts for gastric carcinoma (lymphoma and proximal adenocarcinoma excluded). In the light of 50 papers, this update emphasizes the role of Helicobacter Pylori in gastric carcinogenesis, different staging systems, video-laparoscopic staging, treatment of early and advanced gastric cancer and new biological prognostic factors.     

Activin A and all-trans-retinoic acid cooperatively enhanced the functional activity of L-type Ca2+ channels in the neuroblastoma C1300 cell line

Activin, a member of the transforming growth factor-beta superfamily, regulates various physiological functions. In the present study, we investigated the effect of activin on neuronal differentiation, particularly the functional activity of voltage-dependent Ca2+ channels, in murine neuroblastoma C1300 cells. A slight K(+)-induced increase in the intracellular free Ca2+ ([Ca2+]i) was observed in C1300 cells untreated and treated with either activin A or all-trans-retinoic acid, while treatment with both agents significantly enhanced the increase. The [Ca2+]i increases potentiated by activin A and all-trans-retinoic acid were nearly abolished in the presence of 1.0 mM nickel or in the absence of extracellular Ca2+. Nifedipine (0.1 microM) and omega-conotoxin (1.0 microM), inhibitors of L- and N-type Ca2+ channels, respectively, partially inhibited these responses, however the inhibitory effects of these compounds were not additive. In addition, Bay K 8644, an activator of L-type Ca2+ channels, enhanced the K(+)-induced [Ca2+]i increase. These findings indicated that depolarization evoked the Ca2+ influx, at least in part, through L-type Ca2+ channels in C1300 cells treated with both activin A and all-trans-retinoic acid.     

Chloride channels activated by hypotonicity in N2A neuroblastoma cell line

The acute toxicity of technical-grade glyphosate acid, glyphosate isopropylamine, and three glyphosate formulations was determined for adults of one species and tadpoles of four species of southwestern Australian frogs in 48-h static/renewal tests. The 48-h LC50 values for Roundup(R) Herbicide (MON 2139) tested against tadpoles of Crinia insignifera, Heleioporus eyrei, Limnodynastes dorsalis, and Litoria moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent [AE]), while the 48-h LC50 values for Roundup(R) Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137-144 mg/L (49.4-51.8 mg/L AE). Touchdown(R) Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis, and L. moorei was slightly less toxic than Roundup(R) with 48-h LC50 values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L AE). Roundup(R) Biactive (MON 77920) was practically nontoxic to tadpoles of the same four species producing 48-h LC50 values of 911 mg/L (328 mg/L AE) for L. moorei and >1,000 mg/L (>360mg/L AE) for C. insignifera, H. eyrei, and L. dorsalis. Glyphosate isopropylamine was practically nontoxic, producing no mortality among tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L AE). The toxicity of technical-grade glyphosate acid (48-h LC50, 81.2-121 mg/L) is likely to be due to acid intolerance. Slight differences in species sensitivity were evident, with L. moorei tadpoles showing greater sensitivity than tadpoles of the other four species. Adult and newly emergent metamorphs were less sensitive than tadpoles.     

Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.     

Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.     

Inhibition of M-type K+ and N-type Ca2+ channels by the human gonadotropin-releasing-hormone receptor heterologously expressed in adult neurons

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.     

Biophysical and pharmacological characterization of voltage-dependent Ca2+ channels in neurons isolated from rat nucleus accumbens

The nucleus accumbens (NA) has an integrative role in behavior and may mediate addictive and psychotherapeutic drug action. Whole cell recording techniques were used to characterize electrophysiologically and pharmacologically high- and low-threshold voltage-dependent Ca2+ currents in isolated NA neurons. High-threshold Ca2+ currents, which were found in all neurons studied and include both sustained and inactivating components, activated at potentials greater than -50 mV and reached maximal activation at approximately 0 mV. In contrast, low-threshold Ca2+ currents activated at voltages greater than -64 mV with maximal activation occurring at -30 mV. These were observed in 42% of acutely isolated neurons. Further pharmacological characterization of high-threshold Ca2+ currents was attempted using nimodipine (Nim), omega-conotoxin-GVIA (omega-CgTx) and omega-agatoxin-IVA (omegaAga), which are thought to identify the L, N, and P/Q subtypes of Ca2+ currents, respectively. Nim (5-10 muM) blocked 18%, omegaCgTx (1-2 muM) blocked 25%, and omegaAga (200 nM) blocked 17% of total Ca2+ current. Nim primarily blocked a sustained high-threshold Ca2+ current in a partially reversible manner. In contrast, omegaCgTx irreversibly blocked both sustained and inactivating components. omegaAga irreversibly blocked only a sustained component. In all three of these Ca2+ channel blockers, plus 5 muM omega-conotoxin-MVIIC to eliminate a small unblocked Q-type Ca2+ current (7%), a toxin-resistant high-threshold Ca2+ current remained that was 32% of total Ca2+ current. This current inactivated much more rapidly than the other high-threshold Ca2+ currents, was depressed in 50 muM Ni2+ and reached maximal activation 5-10 mV negative to the toxin-sensitive high-threshold Ca2+ currents. Thus NA neurons have multiple types of high-threshold Ca2+ currents with a large component being the toxin-resistant "R" component.     

Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C

The effects of adrenomedullin (AM), a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2+ concentration ([Ca2+]i) at concentrations greater than 10(-7) M. Human calcitonin gene-related peptide (CGRP), a peptide structurally related to AM, also increased [Ca2+]i with a potency similar to that of AM. AM increased [Ca2+]i in the absence of extracellular Ca2+. Further, AM increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) level in a concentration-dependent manner similar to that of AM-induced [Ca2+]i, suggesting that AM-induced elevation of [Ca2+]i is due to Ca2+ release from Ins(1,4,5)P3-sensitive stores. AM (10(-9) to 10(-6) M) increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the [Ca2+]i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases [Ca2+]i through a mechanism independent of cAMP.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Reduced voltage-dependent Ca2+ signaling in CA1 neurons after brief ischemia in gerbils

An initial overload of intracellular Ca2+ plays a critical role in the delayed death of hippocampal CA1 neurons that die a few days after transient ischemia. Without direct evidence, the prevailing hypothesis has been that Ca2+ overload may recur until cell death. Here, we report the first measurements of intracellular Ca2+ in living CA1 neurons within brain slices prepared 1, 2, and 3 days after transient (5 min) ischemia. With no sign of ongoing Ca2+ overload, voltage-dependent Ca2+ transients were actually reduced after 2-3 days of reperfusion. Resting Ca2+ levels and recovery rate after loading were similar to neurons receiving no ischemic insult. The tetrodotoxin-insensitive Ca spike, normally generated by these neurons, was absent at 2 days postischemia, as was a large fraction of Ca2+-dependent spike train adaptation. These surprising findings may lead to a new perspective on delayed neuronal death and intervention.     

Hair cell-specific splicing of mRNA for the alpha1D subunit of voltage-gated Ca2+ channels in the chicken's cochlea

The L-type voltage-gated Ca2+ channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca2+-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type alpha1D subunit that predominates in hair cells of the chicken's cochlea. The alpha1D subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the alpha1D mRNA contributes to the unusual behavior of the hair cell's voltage-gated Ca2+ channels.     

P2u purinoceptor modulation of intracellular Ca2+ in a human lung adenocarcinoma cell line: down-regulation of Ca2+ influx by protein kinase C

The aim of the present investigation was to study the functional alterations in the stomatognathic system following orthodontic-surgical management of skeletal vertical excess problems. The sample comprised 43 patients who received combined orthodontic-surgical treatment including bilateral vertical ramus osteotomy for posterior repositioning and counterclockwise rotation of the mandible (n = 26) or Le Fort I osteotomy for maxillary impaction (n = 17). All subjects were examined within 1 week before operation and 6 months postsurgery. Methods of examination included: (a) evaluation of dysfunction by means of a clinical index, (b) measurement of mandibular range of motion, (c) assessment of the number and intensity of occlusal contacts, and (d) tomographic evaluation of condyle-fossa relationships. The results of the study indicated that postoperatively (a) there was an increase of patients with dysfunction in the mandibular osteotomy group and a decrease of patients with dysfunction in the maxillary osteotomy group; (b) the maximum interincisal opening decreased significantly in the mandibular osteotomy group; (c) there was a significant increase in the number and intensity of occlusal contacts in both groups; and (d) the shortest posterior and anterior interarticular distances increased significantly in the mandibular osteotomy group.     

The GABAA receptor is expressed in human neurons derived from a teratocarcinoma cell line

NT2 cells, a human teratocarcinoma cell line, are shown to be differentiated in neuron-like cells (NT2-N cells) by treatment with retinoic acid. The present study identified the neurotransmitter receptors expressed in NT2-N cells using patch-clamp recording. Voltage-sensitive Na+ currents, which are specific for neurons, were observed in NT2-N cells but not in NT2 cells, suggesting that NT2-N cells actually function as neurons. Glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, evoked whole-cell currents. In addition, gamma-aminobutyric acid (GABA) evoked currents and the currents were inhibited by the selective GABAA receptor antagonist, bicuculline. In outside-out patches, GABA elicited single channel currents with two classes of the slope conductance (26 and 50 pS). No current, however, was induced by ACh, serotonin, or dopamine NT2-N cells, thus, express at least two types of the major excitatory and inhibitory neurotransmitter receptor in the central nervous system, the glutamate and GAGAA receptors, suggesting that these receptors have a crucial role in neurotransmission from the earlier stage of the brain development.