Slobodan M. Todorovic

Cell-Specific Alterations of T-Type Calcium Current in Painful Diabetic Neuropathy Enhance Excitability of Sensory Neurons

Recent data indicate that T-type Ca2+ channels are amplifiers of peripheral pain signals, but their involvement in disorders of sensory neurons such as those associated with diabetes is poorly understood. To address this issue, we used a combination of behavioral, immunohistological, molecular, and electrophysiological studies in rats with streptozotocin (N-[methylnitrosocarbamoil]-d-glucosamine)-induced early diabetic neuropathy. We found that, in parallel with the development of diabetes-induced pain, T-type current density increased by twofold in medium-size cells from L4–L5 dorsal root ganglia (DRG) with a depolarizing shift in steady-state inactivation. This not only correlated closely with more prominent afterdepolarizing potentials (ADPs) but also increased cellular excitability manifested as a lower threshold for burst firing in diabetic than in control cells. T-type currents and ADPs were potently inhibited by nickel and enhanced by l-cysteine, suggesting that the CaV3.2 T-type channel isoform was upregulated. Both control and diabetic DRG cells with ADPs stained positively for isolectin B4, but only diabetic cells responded robustly to capsaicin, suggesting enhanced nociceptive function. Because increased excitability of sensory neurons may result in such pathological perceptions of pain as hyperalgesia and allodynia, upregulation of T-type Ca2+ currents and enhanced Ca2+ entry into these cells could contribute to the development of symptoms in diabetic neuropathy.

Role of voltage-gated calcium channels in ascending pain pathways

Voltage gated calcium channels (VGCCs) are well established mediators of pain signals in primary afferent neurons. N-type calcium channels are localized to synaptic nerve terminals in laminae 1 and 2 of the dorsal horn where their opening results in the release of neurotransmitters such as glutamate and substance P. The contribution of N-type channels to the processing of pain signals is regulated by alternate splicing of the N-type channel gene, with unique N-type channel splice variants being expressed in small nociceptive neurons. In contrast, T-type VGCCs of the Ca(v)3.2 subtype are likely localized to nerve endings where they regulate cellular excitability. Consequently, inhibition of N-type and Ca(v)3.2 T-type VGCCs has the propensity to mediate analgesia. T-type channel activity is regulated by redox modulation, and can be inhibited by a novel class of small organic blockers. N-type VGCC activity can be potently inhibited by highly selective peptide toxins that are delivered intrathecally, and the search for small organic blockers with clinical efficacy is ongoing. Here, we provide a brief overview of recent advances in this area, as presented at the Spring Pain Research conference (Grand Cayman, 2008).

Upregulation of the T-Type Calcium Current in Small Rat Sensory Neurons After Chronic Constrictive Injury of the Sciatic Nerve

Recent data indicate that peripheral T-type Ca2+ channels are instrumental in supporting acute pain transmission. However, the function of these channels in chronic pain processing is less clear. To address this issue, we studied the expression of T-type Ca2+ currents in small nociceptive dorsal root ganglion (DRG) cells from L4-5 spinal ganglia of adult rats with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. In control rats, whole cell recordings revealed that T-type currents, measured in 10 mM Ba2+ as a charge carrier, were present in moderate density (20 +/- 2 pA/pF). In rats with CCI, T-type current density (30 +/- 3 pA/pF) was significantly increased, but voltage- and time-dependent activation and inactivation kinetics were not significantly different from those in controls. CCI-induced neuropathy did not significantly change the pharmacological sensitivity of T-type current in these cells to nickel. Collectively, our results indicate that CCI-induced neuropathy significantly increases T-type current expression in small DRG neurons. Our finding that T-type currents are upregulated in a CCI model of peripheral neuropathy and earlier pharmacological and molecular studies suggest that T-type channels may be potentially useful therapeutic targets for the treatment of neuropathic pain associated with partial mechanical injury to the sciatic nerve.

Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels

Recent studies have demonstrated an important role for T-type Ca2+ channels (T-channels) in controlling the excitability of peripheral pain-sensing neurons (nociceptors). However, the molecular mechanisms underlying the functions of T-channels in nociceptors are poorly understood. Here, we demonstrate that reducing agents as well as endogenous metal chelators sensitize C-type dorsal root ganglion nociceptors by chelating Zn2+ ions off specific extracellular histidine residues on Cav3.2 T-channels, thus relieving tonic channel inhibition, enhancing Cav3.2 currents, and lowering the threshold for nociceptor excitability in vitro and in vivo. Collectively, these findings describe a novel mechanism of nociceptor sensitization and firmly establish reducing agents, as well as Zn2+, Zn2+-chelating amino acids, and Zn2+-chelating proteins as endogenous modulators of Cav3.2 and nociceptor excitability.

In vivo silencing of the CaV3.2 T-type calcium channels in sensory neurons alleviates hyperalgesia in rats with streptozocin-induced diabetic neuropathy

ABSTRACT Earlier, we showed that streptozocin (STZ)‐induced type 1 diabetes in rats leads to the development of painful peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia and mechanical allodynia accompanied by significant enhancement of T‐type calcium currents (T‐currents) and cellular excitability in medium‐sized dorsal root ganglion (DRG) neurons. Here, we studied the in vivo and in vitro effects of gene‐silencing therapy specific for the CaV3.2 isoform of T‐channels, on thermal and mechanical hypersensitivities, and T‐current expression in small‐ and medium‐sized DRG neurons of STZ‐treated rats. We found that silencing of the T‐channel CaV3.2 isoform using antisense oligonucleotides, had a profound and selective anti‐hyperalgesic effect in diabetic rats and is accompanied by significant down‐regulation of T‐currents in DRG neurons. Anti‐hyperalgesic effects of CaV3.2 antisense oligonucleotides in diabetic rats were similar in models of rapid and slow onset of hyperglycemia following intravenous and intraperitoneal injections of STZ, respectively. Furthermore, treatments of diabetic rats with daily insulin injections reversed T‐current alterations in DRG neurons in parallel with reversal of thermal and mechanical hypersensitivities in vivo. This confirms that CaV3.2 T‐channels, important signal amplifiers in peripheral sensory neurons, may contribute to the cellular hyperexcitability that ultimately leads to the development of painful PDN.

New evidence that both T-type calcium channels and GABAA channels are responsible for the potent peripheral analgesic effects of 5α-reduced neuroactive steroids

&NA; Neurosteroids are potent blockers of neuronal low‐voltage activated (T‐type) Ca2+ channels and potentiators of GABAA ligand‐gated channels, but their effects in peripheral pain pathways have not been studied previously. To investigate potential analgesic effects and the ion channels involved, we tested the ability of locally injected 5α‐reduced neurosteroids to modulate peripheral thermal nociception to radiant heat in adult rats in vivo and to modulate GABAA and T‐type Ca2+ channels in vitro. The steroid anesthetic alphaxalone (ALPX), the endogenous neurosteroid allopregnanolone (3α5αP), and a related compound ((3α,5α,17β)‐3‐hydroxyandrostane‐17‐carbonitrile, (ACN)), induced potent, dose‐dependent, enantioselective anti‐nociception in vivo and modulation of both T‐type Ca2+ currents and GABAA‐mediated currents in vitro. Analgesic effects of ALPX were incompletely antagonized by co‐injections of the GABAA receptor antagonist bicuculline. The neurosteroid analogue ((3α,5α)‐3‐hydroxy‐13,24‐cyclo‐18,21‐dinorchol‐22‐en‐24‐ol (CDNC24), a compound with GABAergic but not T‐type activity, was not analgesic. However, (3β,5α,17β)‐17‐hydroxyestrane‐3‐carbonitrile (ECN)), which has effects on T‐type channels but not on GABAA receptors, also induced potent enantioselective peripheral anti‐nociception. ECN increased pain thresholds less than ALPX, 3α5αP and ACN. However, when an ineffective dose of CDNC24 was combined with ECN, anti‐nociceptive activity was greatly enhanced, and this effect was bicuculline‐sensitive. These results strongly suggest that GABAA channels do not contribute to baseline pain transmission, but they can enhance anti‐nociception mediated by blockade of T‐type Ca2+ channels. In conclusion, we demonstrate that potent peripheral analgesia induced by 5α‐reduced neurosteroid is mediated in part by effects on T‐type Ca2+ channels. Our results also reveal a role of GABA‐gated ion channels in peripheral nociceptive signaling.

T-type voltage-gated calcium channels as targets for the development of novel pain therapies

It is well recognized that voltage‐gated calcium (Ca2+) channels modulate the function of peripheral and central pain pathways by influencing fast synaptic transmission and neuronal excitability. In the past, attention focused on the modulation of different subtypes of high‐voltage‐activated‐type Ca2+ channels; more recently, the function of low‐voltage‐activated or transient (T)‐type Ca2+ channels (T‐channels) in nociception has been well documented. Currently, available pain therapies remain insufficient for certain forms of pain associated with chronic disorders (e.g. neuropathic pain) and often have serious side effects. Hence, the identification of selective and potent inhibitors and modulators of neuronal T‐channels may help greatly in the development of safer, more effective pain therapies. Here, we summarize the available information implicating peripheral and central T‐channels in nociception. We also discuss possible future developments aimed at selective modulation of function of these channels, which are highly expressed in nociceptors.

General Anesthesia Causes Long-term Impairment of Mitochondrial Morphogenesis and Synaptic Transmission in Developing Rat Brain.

Background: Clinically used general anesthetics, alone or in combination, are damaging to the developing mammalian brain. In addition to causing widespread apoptotic neurodegeneration in vulnerable brain regions, exposure to general anesthesia at the peak of synaptogenesis causes learning and memory deficiencies later in life. In vivo rodent studies have suggested that activation of the intrinsic (mitochondria-dependent) apoptotic pathway is the earliest warning sign of neuronal damage, suggesting that a disturbance in mitochondrial integrity and function could be the earliest triggering events. Methods: Because proper and timely mitochondrial morphogenesis is critical for brain development, the authors examined the long-term effects of a commonly used anesthesia combination (isoflurane, nitrous oxide, and midazolam) on the regional distribution, ultrastructural properties, and electron transport chain function of mitochondria, as well as synaptic neurotransmission, in the subiculum of rat pups. Results: This anesthesia, administered at the peak of synaptogenesis, causes protracted injury to mitochondria, including significant enlargement of mitochondria (more than 30%, P < 0.05), impairment of their structural integrity, an approximately 28% increase in their complex IV activity (P < 0.05), and a twofold decrease in their regional distribution in presynaptic neuronal profiles (P < 0.05), where their presence is important for the normal development and functioning of synapses. Consequently, the authors showed that impaired mitochondrial morphogenesis is accompanied by heightened autophagic activity, decrease in mitochondrial density (approximately 27%, P < 0.05), and long-lasting disturbances in inhibitory synaptic neurotransmission. The interrelation of these phenomena remains to be established. Conclusion: Developing mitochondria are exquisitely vulnerable to general anesthesia and may be important early target of anesthesia-induced developmental neurodegeneration.

TTA-P2 is a Potent and Selective Blocker of T-type Calcium Channels in Rat Sensory Neurons and a Novel Antinociceptive Agent

Several agents that are preferential T-type calcium (T-channel) blockers have shown promise as being effective in alleviating acute and chronic pain, suggesting an urgent need to identify even more selective and potent T-channel antagonists. We used small, acutely dissociated dorsal root ganglion (DRG) cells of adult rats to study the in vitro effects of 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), a derivative of 4-aminomethyl-4-fluoropiperdine, on T currents, as well as other currents known to modulate pain transmission. We found that TTA-P2 potently and reversibly blocked DRG T currents with an IC50 of 100 nM and stabilized channel in the inactive state, whereas high-voltage-activated calcium and sodium currents were 100- to 1000-fold less sensitive to channel blocking effects. In in vivo studies, we found that intraperitoneal injections of 5 or 7.5 mg/kg TTA-P2 reduced pain responses in mice in phases 1 and 2 of the formalin test. Furthermore, TTA-P2, at 10 mg/kg i.p., selectively and completely reversed thermal hyperalgesia in diabetic rats treated with streptozocin but had no effect on the nociceptive response of healthy animals. The antihyperalgesic effects of TTA-P2 in diabetic rats were completely abolished by administration of oligonucleotide antisense for CaV3.2 isoform of T channels. Thus, TTA-P2 is not only the most potent and selective blocker of T channels in sensory neurons yet described, but it also demonstrates the potential for the pharmacological effectiveness of this approach in addressing altered nociceptive responses in animal models of both inflammatory and neuropathic pain.

Mechanical and thermal antinociception in rats following systemic administration of mibefradil, a T-type calcium channel blocker.

Voltage-gated Ca(2+) channels play a crucial role in pain perception. We studied the antinociceptive potential of systemically administered mibefradil, a peripherally acting antihypertensive agent and preferential T-type Ca(2+) channel blocker. Systemic injections of mibefradil, in clinically relevant doses, induced mechanical and thermal antinociception in adult rats without compromising their sensorimotor abilities. This suggests that peripherally acting T-type Ca(2+) channel blockers can be useful antinociceptive agents.