Inhibition of T-Type Calcium Channels With TTA-P2 Reduces Chronic Neuropathic Pain Following Spinal Cord Injury in Rats.

Spinal cord injury (SCI)-induced neuropathic pain (SCI-NP) develops in up to 60 to 70% of people affected by traumatic SCI, leading to a major decline in quality of life and increased risk for depression, anxiety, and addiction. Gabapentin and pregabalin, together with antidepressant drugs, are commonly prescribed to treat SCI-NP, but their efficacy is unsatisfactory. The limited efficacy of current pharmacological treatments for SCI-NP likely reflects our limited knowledge of the underlying mechanism(s) responsible for driving the maintenance of SCI-NP. The leading hypothesis in the field supports a major role for spontaneously active injured nociceptors in driving the maintenance of SCI-NP. Recent data from our laboratory provided additional support for this hypothesis and identified the T-type calcium channels as key players in driving the spontaneous activity of SCI-nociceptors, thus providing a rational pharmacological target to treat SCI-NP. To test whether T-type calcium channels contribute to the maintenance of SCI-NP, male and female SCI and sham rats were treated with TTA-P2 (a blocker of T-type calcium channels) to determine its effects on mechanical hypersensitivity (as measured with the von Frey filaments) and spontaneous ongoing pain (as measured with the conditioned place preference paradigm), and compared them to the effects of gabapentin, a blocker of high voltage-activated calcium channels. We found that both TTA-P2 and gabapentin reduced mechanical hypersensitivity in male and females SCI rats, but surprisingly only TTA-P2 reduced spontaneous ongoing pain in male SCI rats. PERSPECTIVES: SCI-induced neuropathic pain, and in particular the spontaneous ongoing pain component, is notoriously very difficult to treat. Our data provide evidence that inhibition of T-type calcium channels reduces spontaneous ongoing pain in SCI rats, supporting a clinically relevant role for T-type channels in the maintenance of SCI-induced neuropathic pain.

Estradiol enhances T-type calcium channel activation in human myometrium telocytes.

Uterine peristalsis is essential for gamete transport and embryo implantation. It shares the characteristics of spontaneity, rhythmicity, and directivity with gastrointestinal peristalsis. Telocytes, the "interstitial Cajal-like cells" outside the digestive canal, are also located in the uterus and may act as pacemakers. To investigate the possible origin and regulatory mechanism of periodic uterine peristalsis in the human menstrual cycle, telocytes in the myometrium were studied to determine the effect of estradiol on T-type calcium channel regulation. In this study, biopsies of the human myometrium were obtained for cell culture, and double-labeling immunofluorescence screening was used to identify telocytes and T-type calcium channel expression. Intracellular calcium signal measurements and patch-clamp recordings were used to investigate the role of T-type calcium channels in regulating calcium currents with or without estradiol. Our study demonstrates that telocytes exist in the human uterus and express T-type calcium channels. The intracellular Ca2+ fluorescence intensity marked by Fluo-4AM was dramatically decreased by NNC 55-0396, a highly selective T-type calcium channel blocker, but enhanced by estradiol. T-type calcium current amplitude increased in telocytes incubated with estradiol in a dose-dependent manner compared to the control group. In conclusion, our study demonstrated that telocytes exist in the human myometrium, expressing T-type calcium channels and estradiol-enhanced T-type calcium currents, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

T-type calcium channel blockade induces apoptosis in C2C12 myotubes and skeletal muscle via endoplasmic reticulum stress activation.

Loss of T-type calcium channel (TCC) function has been reported to result in decreased cell viability and impaired muscle regeneration, but the underlying mechanisms remain largely unknown. We previously found that expression of TCC is reduced in aged pelvic floor muscle of multiple vaginal delivery mice, and this is related to endoplasmic reticulum stress (ERS) activation and autophagy flux blockade. In the present work, we further investigated the effects of TCC function loss on C2C12 myotubes and skeletal muscle, which is mediated by promotion of ERS and ultimately contributes to mitochondrial-related apoptotic cell death. We found that application of a TCC inhibitor induced mitochondria-related apoptosis in a dose-dependent manner and also reduced mitochondrial transmembrane potential (MMP), induced mito-ROS generation, and enhanced expression of mitochondrial apoptosis proteins. Functional inhibition of TCC induced ERS, resulting in disorder of Ca2+ homeostasis in endoplasmic reticulum, and ultimately leading to cell apoptosis in C2C12 myotubes. Tibialis anterior muscles of T-type α1H channel knockout mice displayed a smaller skeletal muscle fiber size and elevated ERS-mediated apoptosis signaling. Our data point to a novel mechanism whereby TCC blockade leads to ERS activation and terminal mitochondrial-related apoptotic events in C2C12 myotubes and skeletal muscles.

The prostaglandin E2/EP4 receptor/cyclic AMP/T-type Ca(2+) channel pathway mediates neuritogenesis in sensory neuron-like ND7/23 cells.

We investigated mechanisms for the neuritogenesis caused by prostaglandin E2 (PGE2) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE2 caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Cav3.1 and Cav3.2 T-type Ca(2+) channels (T-channels). The neuritogenesis induced by db-cAMP or PGE2 was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE2/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons.

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential.

Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type Ca(V)1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (Ca(V)3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (Ca(V)2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., Ca(V)1.2 and Ca(V)1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective Ca(V)1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson's disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

Evidence for postsynaptic modulation of muscle contraction by a Drosophila neuropeptide.

DPKQDFMRFamide, the most abundant FMRFamide-like peptide in Drosophila melanogaster, has been shown previously to enhance contractions of larval body wall muscles elicited by nerve stimulation and to increase excitatory junction potentials (EJPs). The present work investigated the possibility that this peptide can also stimulate muscle contraction by a direct action on muscle fibers. DPKQDFMRFamide induced slow contractions and increased tonus in body wall muscles of Drosophila larvae from which the central nervous system had been removed. The threshold for this effect was approximately 10(-8)M. The increase in tonus persisted in the presence of 7x10(-3)M glutamate, which desensitized postsynaptic glutamate receptors. Thus, the effect on tonus could not be explained by enhanced release of glutamate from synaptic terminals and, thus, may represent a postsynaptic effect. The effect on tonus was abolished in calcium-free saline and by treatment with L-type calcium channel blockers, nifedipine and nicardipine, but not by T-type blockers, amiloride and flunarizine. The present results provide evidence that this Drosophila peptide can act postsynaptically in addition to its apparent presynaptic effects, and that the postsynaptic effect requires influx through L-type calcium channels.

Ligand-based virtual screening to identify new T-type calcium channel blockers.

T-type calcium channels are involved in the generation of rhythmical firing patterns in the mammalian central nervous system and in various pathological alterations of neuronal excitability such as in epilepsy or neuropathic pain. In the search for new T-type calcium channel blockers that would help to treat these disorders, we have followed a bi-dimensional pharmacophore-based virtual screening approach to identify new inhibitors. Nineteen molecules extracted from AurSCOPE Ion Channels knowledgebase were used as query molecules to screen an external database. This in silico approach was then validated using electrophysiology. Interestingly, 16 compounds out of 38 distinct molecules tested showed more than 50% blockade of the Ca(V)3.2 mediated T-type current. Two series of compounds show chemical originality compared with known T-type calcium channel blockers.

Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion.

The hypothesis that Ca2+ influx necessary for angiotensin II (AngII) and K+ stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential (Vm) demonstrated that AngII and K+ induce concentration-dependent depolarizations capable of activating T channels and, at high K+ and AngII concentrations, activating L channels and inactivating T channels. K+-induced depolarizations were stable and readily reversible. Vm was proportional to K+ concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K+. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in Vm were occasionally observed in response to 10(-9) M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K+ conductance. Neither rapid fluctuations nor slow oscillations in Vm were blocked by mibefradil or other treatments that inhibit voltage-gated Ca2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10(-8) M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K+-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K(app) of 0.62 microM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K+- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 microM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K+-induced secretion. The IC50 for K+-induced secretion was dependent on K+ concentration, increasing from 0.2 microM for 6 mM K+ to 2.5 microM for 10 mM K+ or greater. Mibefradil exhibited an IC50 of 1.1 microM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K+ conductances, inhibition of Ca2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K+). In summary, these results indicate that voltage-gated T channels represent the primary Ca2+ influx pathway activated by physiological concentrations of AngII and K+ but other Ca2+ influx pathways must mediate aldosterone secretion induced by high K+ or AngII concentrations.