Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 µM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α1G, α1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.     

Prostanoid-dependent bladder pain caused by proteinase-activated receptor-2 activation in mice: Involvement of TRPV1 and T-type Ca2+ channels

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely ‘referred hyperalgesia’, 6–24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca²⁺ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E₂ production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca²⁺ channels.     

Cardiomyocyte Ca<Superscript>2+</Superscript> overload in atrial tachycardia: is blockade of L-type Ca<Superscript>2+</Superscript> channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca²⁺ overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca²⁺ overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca²⁺ channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca²⁺ channel current (I_Ca,L) that may reinforce Ca²⁺ overload thereby promoting AF in the atrium. Ca²⁺ channel blockers which target T-type Ca²⁺ channels in addition to I_Ca,L (for instance, efonidipine) may be more efficient at preventing Ca²⁺ overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca²⁺ channel blockers that additionally target T-type Ca²⁺ channels.     

The toxic effects of Bisphenol A on the mouse spermatocyte GC‐2 cell line: the role of the Ca2+‐calmodulin‐Ca2+/calmodulin‐dependent protein kinase II axis

Bisphenol A (BPA), an endocrine‐disrupting chemical (EDC), is known to induce male reproductive toxicity in rodents. However, its toxic effects on the germ cells are still poorly understood. It has been proposed that Ca²⁺ homeostasis and Ca²⁺ sensors, including calmodulin (CaM) and calmodulin‐dependent protein kinase II (CaMKII), play critical roles in spermatogenesis. Therefore, in the present study, we aimed to investigate whether a perturbation in Ca²⁺‐CaM‐CaMKII signaling was involved in the BPA‐induced injury to mouse spermatocyte GC‐2spd (ts) (GC‐2) cells. Our results showed that BPA (range from 0.2 to 20 μM) induced obvious GC‐2 cell injury, including decreased cell viability, the release of mitochondrial cytochrome c and the activation of caspase‐3. However, these processes could be partially abrogated by pretreatment with a Ca²⁺ chelator (BAPTA/AM), a CaM antagonist (W7) or a CaMKII inhibitor (KN93). These results, taken together, indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca²⁺/CaM/CaMKII signaling and the mitochondrial apoptotic process. Copyright © 2015 John Wiley & Sons, Ltd.     

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes

Cardiac hypertrophy impairs Ca²⁺ handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca²⁺ release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca²⁺-dependent signaling and the phosphorylation of phospholamban, which regulates Ca²⁺ uptake during myocardial relaxation and is in turn regulated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72 h and remained elevated for at least 96 h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca²⁺ mobilization all peaked 48 h after these treatments. By 96 h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca²⁺ mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca²⁺ regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

Tarantula Toxin ProTx-I Differentiates Between Human T-type Voltage-Gated Ca2+ Channels Cav3.1 and Cav3.2

ProTx-I peptide, a venom toxin of the tarantula Thrixopelma pruriens, has been reported to interact with voltage-gated ion channels. ProTx-I reduced Ba²⁺ currents through recombinant human T-type voltage-gated Ca²⁺ channels, Ca_v3.1 (hCa_v3.1), with roughly 160-fold more potency than through hCa_v3.2 channels. Chimeric channel proteins (hCa_v3.1/S3S4 and hCa_v3.2/ S3S4) were produced by exchanging fourteen amino acids in the hCa_v3.1 domain IV S3-S4 linker region and the corresponding region of hCa_v3.2 between each other. The ProTx-I sensitivity was markedly reduced in the hCa_v3.1/S3S4 chimera as compared to the original hCa_v3.1 channel, while the hCa_v3.2/S3S4 chimera exhibited greater ProTx-I sensitivity than the original hCa_v3.2 channel. These results suggest that the domain IV S3-S4 linker in the hCa_v3.1 channel may contain residues involved in the interaction of ProTx-I with T-type Ca²⁺ channels.     

Ca2+/calmodulin dependent kinase II: A critical mediator in determining reperfusion outcomes in the heart?

Ischaemic heart disease is a major cause of death and disability in the Western world, and a substantial health burden. Cardiomyocyte Ca²⁺ overload is known to significantly contribute to contractile dysfunction and myocyte death in ischaemia and reperfusion, and significant advancements have been made in identifying the downstream mediators and cellular origins of this Ca²⁺ mismanagement. Ca²⁺/calmodulin‐dependent kinase II (CaMKII) is recognized as an important mediator linking pathological changes in subcellular environments to modifications in cardiomyocyte Ca²⁺ handling. Activated in response to fluctuations in cellular Ca²⁺ and to various post‐translational modifications, CaMKII targets numerous Ca²⁺ channels/transporters involved in Ca²⁺ handling and contractile function regulation. CaMKII is activated early in reperfusion, where it exacerbates Ca²⁺ leak from the sarcoplasmic reticulum and promotes the onset of ventricular arrhythmias. Inhibiting CaMKII can increase functional recovery in reperfusion and reduce apoptotic/necrotic death, at least partly through indirect and direct influences on mitochondrial Ca²⁺ levels and function. Yet, CaMKII can also have beneficial actions in ischaemia and reperfusion, in part by providing inotropic support for the stunned myocardium and contributing as an intermediate to cardioprotective preconditioning signalling cascades. There is considerable potential in targeting CaMKII as a part of a surgical reperfusion strategy, though further mechanistic understanding of the relationship between CaMKII activation status and the extent of ischaemia/reperfusion injury are required to fully establish an optimal pharmacological approach.     

Br-DIF-1 accelerates dimethyl sulphoxide-induced differentiation of P19CL6 embryonic carcinoma cells into cardiomyocytes

BACKGROUND AND PURPOSE

Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells.

EXPERIMENTAL APPROACH

P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca²⁺ channel blocker.

KEY RESULTS

Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3–3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca²⁺ channel blocker; 100 nM) and a small interfering RNA for the T-type Ca²⁺ channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO.

CONCLUSIONS AND IMPLICATIONS

Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca²⁺ channel gene.     

SELECTIVITY OF DIFFERENT CALCIUM ANTAGONISTS ON T- AND L-TYPE CALCIUM CURRENTS IN GUINEA-PIG VENTRICULAR MYOCYTES

Both L- and T-type calcium channels are present in the heart. In cardiac myocytes L-type calcium channels are blocked by the classical calcium channel blockers, while T-type calcium channels are thought to be insensitive to these drugs and to be selectively blocked by mibefradil. We aimed to compare the T/L calcium channel blocking selectivity of several calcium channel blockers by evaluating their effects on both components evoked in the same cell from a holding potential corresponding to the normal physiological value (−90 mV). Currents were recorded in single patch-clamped guinea-pig ventricular myocytes, superfused with a Na⁺- and K⁺-free solution to abolish overlapping currents. Two dihydropyridines (amlodipine and lacidipine), verapamil diltiazem and mibefradil were tested; for each compound concentrations equieffective on L-type Ca²⁺ current were used. All calcium channel blockers, at concentrations blocking less than 30% of L-type Ca²⁺ current, inhibited a significant amount of T-type Ca²⁺ current, varying from 0.8% (diltiazem) to 28% (mibefradil). We calculated for each compound the T/L ratio. As expected, mibefradil showed the highest T selectivity; lacidipine and diltiazem resulted to be L selective. Verapamil and amlodipine were not selective. Thus, the calcium channel blockers can be differentiated on the basis of their T/L selectivity.     

CaMKIIδ and cardiomyocyte Ca2+ signalling new perspectives on splice variant targeting

Control of cardiomyocyte cytosolic Ca²⁺ levels is crucial in determining inotropic status and ischemia/reperfusion stress response. Responsive to fluctuations in cellular Ca²⁺, Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is a serine/threonine kinase integral to the processes regulating cardiomyocyte Ca²⁺ channels/transporters. CaMKII is primarily expressed either in the δ_B or δ_C splice variant forms, which may mediate differential influences on cardiomyocyte function and pathological response mechanisms. Increases in myocyte Ca²⁺ levels promote the binding of a Ca²⁺/calmodulin complex to CaMKII, to activate the kinase. Activity is also maintained through a series of post‐translational modifications within a critical region of the regulatory domain of the protein. Recent data indicate that the post‐translational modification status of CaMKIIδ_B/δ_C variants may have an important influence on reperfusion outcomes. This study provided the first evidence that the specific type of CaMKII post‐translational modification has a role in determining target selectivity of downstream Ca²⁺ transporters. The study was also able to demonstrate that the phosphorylated form of CaMKII closely co‐localizes with CaMKIIδ_B in the nuclear/myofilament fraction, contrasting with a co‐enrichment of oxidized CaMKII in the membrane fraction with CaMKIIδ_C. It has also been possible to conclude that a hyper‐phosphorylation of CaMKII (Thr287) in reperfused hearts represents a hyper‐activation of the CaMKIIδ_B, which exerts anti‐arrhythmic actions through an enhanced capacity to selectively increase sarcoplasmic reticulum Ca²⁺ uptake and maintain cytosolic Ca²⁺ levels. This suggests that suppression of global CaMKIIδ may not be an efficacious approach to developing optimal pharmacological interventions for the vulnerable heart.     

BAFF activates Erk1/2 promoting cell proliferation and survival by Ca-CaMKII-dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca²⁺-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca²⁺-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca²⁺ may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.     

Role of calcium dysregulation in Alzheimer's disease and its therapeutic implications

The increasing incidence of Alzheimer's disease (AD) coupled with the lack of therapeutics to address the underlying pathology of the disease has necessitated the need for exploring newer targets. Calcium dysregulation represents a relatively newer target associated with AD. Ca⁺² serves as an important cellular messenger in neurons. The concentration of the Ca⁺² ion needs to be regulated at optimal concentrations intracellularly for normal functioning of the neurons. This is achieved with the help of mitochondria, endoplasmic reticulum, and neuronal plasma membrane channel proteins. Disruption in normal calcium homeostasis can induce formation of amyloid beta plaques, accumulation of neurofibrillary tangles, and dysfunction of synaptic plasticity, which in turn can affect calcium homeostasis further, thus forming a vicious cycle. Hence, understanding calcium dysregulation can prove to be a key to develop newer therapeutics. This review provides detailed account of physiology of calcium homeostasis and its dysregulation associated with AD. Further, with an understanding of various receptors and organelles involved in these pathways, the review also discusses various calcium channel blockers explored in AD hand in hand with some multitarget molecules addressing calcium as one of the targets.     

Changes of cardiac calcium homeostasis in spontaneously hypertensive rats

1 The aim of the present study was to assess the alterations in cardiac Ca²⁺ homeostasis induced by hypertension using electrically paced right ventricular strips from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2 Basal contractile force was higher in SHR than in WKY. Similarly, the β-adrenoceptor agonist isoprenaline (10 n m –10 μm ) induced a concentration-dependent positive inotropic effect that was higher in SHR than in WKY, which was in turn inhibited by the β-adrenoceptor antagonist propranolol (1 μm ) in both strains. 3 Preincubation of strips with the L-type Ca²⁺ channel blockers, nifedipine (1 μm ) or verapamil (10 μm ), markedly inhibited the isoprenaline response, the inhibition being higher in SHR than in WKY. However, this inhibition was minor by the T-type Ca²⁺ channel blocker mibefradil (10 μm ). 4 Bay K 8644 (10 n m –10 μm ), a L-type Ca²⁺ channel activator induced a concentration-dependent positive inotropic effect, that was greater in SHR than WKY. 5 Nifedipine and verapamil (both 0.1 n m –10 μm ) inhibited in a concentration-dependent way the inotropic effect induced by 0.3 μm isoprenaline or 1 μm Bay K 8644. The inhibition was higher in SHR than in WKY. Mibefradil (0.1 n m –10 μm ) only clearly inhibited the isoprenaline and Bay K 8644 inotropic effects at 10 μm in both strains. 6 The inhibitor of the sarcoplasmic reticulum Ca²⁺ release, ryanodine (10 n m –10 μm ), was a more effective depressor of isoprenaline-induced response in SHR than in WKY. 7 These results suggest that cardiac Ca²⁺ homeostasis in SHR ventricular strips is altered compared with those of WKY, showing an increased Ca²⁺ entry through L-type Ca²⁺ channels and release from sarcoplasmic reticulum; the participation of T-type Ca²⁺ channels are irrelevant in this tissue.     

Investigational calcium channel blockers for the treatment of hypertension

Introduction: Voltage-gated Ca²⁺ channels are the primary route of Ca²⁺ entry in vascular smooth muscle cells, playing a key role in the regulation of arterial tone and blood pressure. Since the 60´s, L-type Ca²⁺ channel blockers (CCBs) have been widely used for the treatment of hypertension.

Areas covered: T-type Ca²⁺ channels regulate vascular tone in small-resistance vessels and aldosterone secretion, and N-type channels expressed in sympathetic nerve terminals regulate the release of neurotransmitters. We performed a literature search in MEDLINE, PubMed and ClinicalTrials.gov to identify eligible studies published between January 2001 and March 2016 and reviewed the antihypertensive and renoprotective effects of four CCBs with different pharmacological profiles: azelnidipine (L-type), cilnidipine (L-/N-type) and benidipine and efonidipine (L-/T-type CCBs). Despite similar blood pressure lowering effects, L/N- and L/T-type CCBs, compared with L-type CCBs, decreased intraglomerular pressure, improved renal hemodynamics and provided a greater decrease in proteinuria even in patients already treated with renin-angiotensin-aldosterone inhibitors.

Expert opinion: Dual L/N- and L/T-type CCBs may exhibit therapeutic advantages over L-type blockers in hypertensive patients with chronic kidney disease. Because clinical trials supporting these advantages present important biases, further large-scale, long-term comparative trials are needed to confirm that these differences translate into improved clinical outcomes.     

Inhibition of T-type Ca²⁺ channels by endostatin attenuates human glioblastoma cell proliferation and migration

BACKGROUND AND PURPOSE

Endostatin (ES) is a c-terminal proteolytic fragment of collagen XVIII with promising antitumour properties in several tumour models, including human glioblastoma. We hypothesized that this peptide could interact with plasma membrane ion channels and modulate their functions.

EXPERIMENTAL APPROACH

Using cell proliferation and migration assays, patch clamp and Western blot analysis, we studied the effects of ES on the proliferation and migration of human glioblastoma U87 cells, mediated by T-type Ca²⁺ channels.

KEY RESULTS

Extracellular application of ES reversibly inhibited T-type Ca²⁺ channel currents (T-currents) in U87 cells, whereas L-type Ca²⁺ currents were not affected. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation but was independent of G-protein and protein tyrosine kinase-mediated pathways. All three α₁ subunits of T-type Ca²⁺ channels (Ca_V3), α_1G (Ca_V3.1), α_1H (Ca_V3.2) and α_1I (Ca_V3.3), were endogenously expressed in U87 cells. Using transfected HEK293 or CHO cells, we showed that only Ca_V3.1 and Ca_V3.2, but not Ca_V3.3 or Ca_V1.2 (L-type), channel currents were significantly inhibited. More interestingly, ES inhibited the proliferation and migration of U87 cells in a dose-dependent manner. Pretreatment of the cells with the specific T-type Ca²⁺ channel blocker mibefradil occluded these inhibitory effects of ES.

CONCLUSION AND IMPLICATIONS

This study provides the first evidence that the antitumour effects of ES on glioblastoma cells is through direct inhibition of T-type Ca²⁺ channels and gives new insights into the future development of a new class of antiglioblastoma agents that target the proliferation and migration of these cells.

LINKED ARTICLE

This article is commented on by Santoni et al., pp. 1244–1246 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01908.x     

Antagonism of Neuronal Prostaglandin E2 Receptor Subtype 1 Mitigates Amyloid β Neurotoxicity In Vitro

Multiple lines of evidence indicate that regional brain eicosanoid signaling is important in initiation and progression of neurodegenerative conditions that have neuroinflammatory pathologic component, such as AD. We hypothesized that PGE₂ receptor subtype 1 (EP1) signaling (linked to intracellular Ca²⁺ release) regulates Aβ peptide neurotoxicity and tested this in two complementary in vitro models: a human neuroblastoma cell line (MC65) producing Aβ_1–40 through conditional expression of the APP C-terminal portion, and murine primary cortical neuron cultures exposed to Aβ_1–42. In MC65 cells, EP1 receptor antagonist SC-51089 reduced Aβ neurotoxicity ~50 % without altering high molecular weight Aβ immunoreactive species formation. Inositol-3-phosphate receptor antagonist 2-aminoethoxy-diphenyl borate offered similar protection. SC-51089 largely protected the neuron cultures from synthetic Aβ_1–42 neurotoxicity. Nimodipine, a Ca²⁺ channel blocker, was completely neuroprotective in both models. Based on these data, we conclude that suppressing neuronal EP1 signaling may represent a promising therapeutic approach to ameliorate Aβ peptide neurotoxicity.     

Role of T-type Ca2+ channel inhibitors in the pacemaker depolarization in rabbit sino-atrial nodal cells

• 1.1. Effects of T-type Ca²⁺ channel inhibitors, Ni²⁺ and tetramethrin, on the spontaneous action potentials in rabbit sino-atrial nodal cells were examined.
• 2.2. The firing rate of spontaneous activity was 201 ± 11 beats/min (n = 18). Experiments were performed at 36°C.
• 3.3. Ni²⁺ (10⁻⁵ to 10⁻⁴ M) and tetramethrin (10⁻⁷ to 5 × 10⁻⁵M) caused a negative chronotropic effect. Both inhibitors did not affect the maximum diastolic potential, and slowed the rate of depolarization during the diastole.
• 4.4. In the presence of TTX (10⁻⁷ M), both inhibitors caused a stronger negative chronotropic effect, and hyperpolarized the maximum diastolic potential. The maximum rate of depolarization was enhanced, and the action potential duration (at 50% repolarization) was prolonged. The action potential amplitude was unaffected. Ni²⁺ had more potent actions than tetramethrin.
• 5.5. T-type and other Ca²⁺ channel inhibitors affected only the late phase of pacemaker potential, resulting in a negative chronotropic effect.
• 6.6. These results indicate that T-type Ca²⁺ channel inhibitors (Ni²⁺ and tetramethrin) slow the pacemaker depolarization at the late phase (but not at the initial phase), resulting in a negative chronotropic effect in the sino-atrial nodal cells.

     

Phasic and Tonic Inhibition are Maintained Respectively by CaMKII and PKA in the Rat Visual Cortex

Phasic and tonic γ-aminobutyric acid_A (GABA_A) receptor-mediated inhibition critically regulate neuronal information processing. As these two inhibitory modalities have distinctive features in their receptor composition, subcellular localization of receptors, and the timing of receptor activation, it has been thought that they might exert distinct roles, if not completely separable, in the regulation of neuronal function. Inhibition should be maintained and regulated depending on changes in network activity, since maintenance of excitation-inhibition balance is essential for proper functioning of the nervous system. In the present study, we investigated how phasic and tonic inhibition are maintained and regulated by different signaling cascades. Inhibitory postsynaptic currents were measured as either electrically evoked events or spontaneous events to investigate regulation of phasic inhibition in layer 2/3 pyramidal neurons of the rat visual cortex. Tonic inhibition was assessed as changes in holding currents by the application of the GABA_A receptor blocker bicuculline. Basal tone of phasic inhibition was maintained by intracellular Ca²⁺ and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). However, maintenance of tonic inhibition relied on protein kinase A activity. Depolarization of membrane potential (5 min of 0 mV holding) potentiated phasic inhibition via Ca²⁺ and CaMKII but tonic inhibition was not affected. Thus, phasic and tonic inhibition seem to be independently maintained and regulated by different signaling cascades in the same cell. These results suggest that neuromodulatory signals might differentially regulate phasic and tonic inhibition in response to changes in brain states.     

Key mechanisms underlying netrin‐1 prevention of impaired spatial and object memory in Aβ1‐42 CA1‐injected rats

Alzheimer's disease (AD) is a neurodegenerative disorder with an incompletely defined aetiology that is associated with memory and cognitive impairment. Currently available therapeutics only provide temporary assistance with symptoms. In spite of plentiful research in the field and the generation of thousands of studies, much is still to be clarified on precise mechanisms of pathobiology, prevention modalities, disease course and cure. Netrin‐1, a laminin family protein, is said to have anti‐inflammatory and anti‐apoptotic effects and has a key role in neurogenesis and morphogenesis of neural structures. Accordingly, this study was designed to investigate protective effects of bilateral intrahippocampal fissure microinjections of netrin‐1 on memory impairment in rat model of AD. Concomitant administration of netrin‐1 with amyloid beta 1‐42 (Aβ_1‐42) improved cognitive dysfunction in novel object recognition task (NOR), ameliorated impaired spatial memory in Morris water maze (MWM) setting, increased neuronal density and reduced amyloid aggregation in rat AD model. Netrin‐1 was also seen to prevent Aβ_1‐42‐induced caspase‐3, caspase‐7 and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) activation. Therefore, based on the data reported here, netrin‐1 may be a promising biologic therapeutic that addresses the memory and neuronal loss associated with AD.