Madhavi Latha Yadav Bangaru

Store-Operated Ca2+ Entry in Sensory Neurons: Functional Role and the Effect of Painful Nerve Injury

Painful nerve injury disrupts levels of cytoplasmic and stored Ca2+ in sensory neurons. Since influx of Ca2+ may occur through store-operated Ca2+ entry (SOCE) as well as voltage- and ligand-activated pathways, we sought confirmation of SOCE in sensory neurons from adult rats and examined whether dysfunction of SOCE is a possible pathogenic mechanism. Dorsal root ganglion neurons displayed a fall in resting cytoplasmic Ca2+ concentration when bath Ca2+ was withdrawn, and a subsequent elevation of cytoplasmic Ca2+ concentration (40 ± 5 nm) when Ca2+ was reintroduced, which was amplified by store depletion with thapsigargin (1 μm), and was significantly reduced by blockers of SOCE, but was unaffected by antagonists of voltage-gated membrane Ca2+ channels. We identified the underlying inwardly rectifying Ca2+-dependent ICRAC (Ca2+ release activated current), as well as a large thapsigargin-sensitive inward current activated by withdrawal of bath divalent cations, representing SOCE. Molecular components of SOCE, specifically STIM1 and Orai1, were confirmed in sensory neurons at both the transcript and protein levels. Axonal injury by spinal nerve ligation (SNL) elevated SOCE and ICRAC. However, SOCE was comparable in injured and control neurons when stores were maximally depleted by thapsigargin, and STIM1 and Orai1 levels were not altered by SNL, showing that upregulation of SOCE after SNL is driven by store depletion. Blockade of SOCE increased neuronal excitability in control and injured neurons, whereas injured neurons showed particular dependence on SOCE for maintaining levels of cytoplasmic and stored Ca2+, which indicates a compensatory role for SOCE after injury.

Failure of action potential propagation in sensory neurons: mechanisms and loss of afferent filtering in C-type units after painful nerve injury

The peripheral terminals of sensory neurons encode physical and chemical signals into trains of action potentials (APs) and transmit these trains to the CNS. Although modulation of this process is thought to predominantly reside at synapses, there are also indications that AP trains are incompletely propagated past points at which axons branch. One such site is the T‐junction, where the single sensory neuron axon branches into peripheral and central processes. In recordings from sensory neurons of dorsal root ganglia excised from adult rats, we identified use‐dependent failure of AP propagation between the peripheral and central processes that results in filtering of rapid AP trains, especially in C‐type neurons. Propagation failure was regulated by membrane input resistance and Ca2+‐sensitive K+ and Cl− currents. Following peripheral nerve injury, T‐junction filtering is reduced in C‐type neurons, which may possibly contribute to pain generation.

Painful nerve injury increases plasma membrane Ca2+-ATPase activity in axotomized sensory neurons

BackgroundThe plasma membrane Ca2+-ATPase (PMCA) is the principal means by which sensory neurons expel Ca2+ and thereby regulate the concentration of cytoplasmic Ca2+ and the processes controlled by this critical second messenger. We have previously found that painful nerve injury decreases resting cytoplasmic Ca2+ levels and activity-induced cytoplasmic Ca2+ accumulation in axotomized sensory neurons. Here we examine the contribution of PMCA after nerve injury in a rat model of neuropathic pain.ResultsPMCA function was isolated in dissociated sensory neurons by blocking intracellular Ca2+ sequestration with thapsigargin, and cytoplasmic Ca2+ concentration was recorded with Fura-2 fluorometry. Compared to control neurons, the rate at which depolarization-induced Ca2+ transients resolved was increased in axotomized neurons after spinal nerve ligation, indicating accelerated PMCA function. Electrophysiological recordings showed that blockade of PMCA by vanadate prolonged the action potential afterhyperpolarization, and also decreased the rate at which neurons could fire repetitively.ConclusionWe found that PMCA function is elevated in axotomized sensory neurons, which contributes to neuronal hyperexcitability. Accelerated PMCA function in the primary sensory neuron may contribute to the generation of neuropathic pain, and thus its modulation could provide a new pathway for peripheral treatment of post-traumatic neuropathic pain.

Ca2+-Dependent Regulation of Ca2+ Currents in Rat Primary Afferent Neurons: Role of CaMKII and the Effect of Injury

Currents through voltage-gated Ca2+ channels (ICa) may be regulated by cytoplasmic Ca2+ levels ([Ca2+]c), producing Ca2+-dependent inactivation (CDI) or facilitation (CDF). Since ICa regulates sensory neuron excitability, altered CDI or CDF could contribute to pain generation after peripheral nerve injury. We explored this by manipulating [Ca2+]c while recording ICa in rat sensory neurons. In uninjured neurons, elevating [Ca2+]c with a conditioning prepulse (−15 mV, 2 s) inactivated ICa measured during subsequent test pulses (−15 mV, 5 ms). This inactivation was Ca2+-dependent (CDI), since it was decreased with elimination of Ca2+ influx by depolarization to above the ICa reversal potential, with high intracellular Ca2+ buffering (EGTA 10 mm or BAPTA 20 mm), and with substitution of Ba2+ for extracellular Ca2+, revealing a residual voltage-dependent inactivation. At longer latencies after conditioning (>6 s), ICa recovered beyond baseline. This facilitation also proved to be Ca2+-dependent (CDF) using the protocols limiting cytoplasmic Ca2+ elevation. Ca2+/calmodulin-dependent protein kinase II (CaMKII) blockers applied by bath (KN-93, myristoyl-AIP) or expressed selectively in the sensory neurons (AIP) reduced CDF, unlike their inactive analogues. Protein kinase C inhibition (chelerythrine) had no effect. Selective blockade of N-type Ca2+ channels eliminated CDF, whereas L-type channel blockade had no effect. Following nerve injury, CDI was unaffected, but CDF was eliminated in axotomized neurons. Excitability of sensory neurons in intact ganglia from control animals was diminished after a similar conditioning pulse, but this regulation was eliminated by injury. These findings indicate that ICa in sensory neurons is subject to both CDI and CDF, and that hyperexcitability following injury-induced loss of CDF may result from diminished CaMKII activity.

Selective Estrogen Receptor Down-Regulator and Selective Estrogen Receptor Modulators Differentially Regulate Lactotroph Proliferation

Background We recently reported that estrogen receptor α (ERα), even in absence of estrogen (E2), plays a critical role in lactotroph homeostasis. The anti-estrogen ICI 182780 (ICI), but not tamoxifen or raloxifene, rapidly promoted the degradation of ERα, and inhibited cell proliferation. However, all three ER antagonists suppressed PRL release, suggesting that receptor occupation is sufficient to inhibit prl gene expression whereas receptor degradation is required to suppress lactotroph proliferation. In this study our objective was to determine whether ERα degradation versus occupation, differentially modulates the biological outcome of anti-estrogens. Principal Findings Using the rat lactotroph cell line, GH3 cells, we report that ICI induced proteosome mediated degradation of ERα. In contrast, an ERα specific antagonist, MPP, that does not promote degradation of ERα, did not inhibit cell proliferation. Further, ICI, but not MPP, abolished anchorage independent growth of GH3 cells. Yet, both ICI and MPP were equally effective in suppressing prl expression and release, as well as ERE-mediated transcriptional activity. Conclusion Taken together, our results demonstrate that in lactotrophs, ERα degradation results in decreased cell proliferation, whereas ERα occupation by an antagonist that does not promote degradation of ERα is sufficient to inhibit prl expression.

Growth Suppression of Mouse Pituitary Corticotroph Tumor AtT20 Cells by Curcumin: A Model for Treating Cushing's Disease

Background Pituitary corticotroph tumors secrete excess adrenocorticotrophic hormone (ACTH) resulting in Cushings disease (CD). Standard treatment includes surgery and, if not successful, radiotherapy, both of which have undesirable side effects and frequent recurrence of the tumor. Pharmacotherapy using PPARγ agonists, dopamine receptor agonists, retinoic acid or somatostatin analogs is still experimental. Curcumin, a commonly used food additive in South Asian cooking, has potent growth inhibitory effects on cell proliferation. Our laboratory recently demonstrated that curcumin inhibited growth and induced apoptosis in prolactin- and growth hormone-producing tumor cells [1]. Subsequently, Schaaf et.al. confirmed our findings and also showed the in vivo effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown. Principal Findings Using the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFκB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells. Conclusion The ability of curcumin to inhibit NFκB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD.

Epidermal growth factor receptor cross-talks with ligand-occupied estrogen receptor-α to modulate both lactotroph proliferation and prolactin gene expression

Both estrogen (E2) and EGF regulate lactotrophs, and we recently demonstrated that EGF phosphorylates S118 on estrogen receptor-alpha (ERalpha) and requires ERalpha to stimulate prolactin (PRL) release. However, the interactions between ligand-occupied ERalpha and activated ErbB1 and its impact on lactotroph function are unknown. Using rat GH3 lactotrophs, we found that both E2 and EGF independently stimulated proliferation and PRL gene expression. Furthermore, their combination resulted in an enhanced stimulatory effect on both cell proliferation and PRL gene expression. Inhibitors of ER as well as ErbB1 blocked the combined effects of E2 and EGF. Pretreatment with UO126 abolished the combined effects, demonstrating Erk1/2 requirement. Although bidirectionality in ER-ErbB1 cross-talk is a well-accepted paradigm, interestingly in lactotrophs, ErbB1 kinase inhibitor failed to block the effect of E2 on proliferation and stimulation of PRL gene expression, suggesting that ER does not require ErbB1 to mediate its effects. Furthermore, E2 did not affect the ability of EGF to induce c-Fos expression or modulate AP-1 activity. However, both E2 and EGF combine to enhance S118 phosphorylation of ERalpha, leading to enhanced E2-mediated estrogen response element transactivation. Taken together, our results suggest that, in lactotrophs, activated ErbB1 phosphorylates ERalpha to enhance the stimulatory effect of E2, thereby providing the molecular basis by which EGF amplifies the response of E2.

Differential expression of CaMKII isoforms and overall kinase activity in rat dorsal root ganglia after injury

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) decodes neuronal activity by translating cytoplasmic Ca(2+) signals into kinase activity that regulates neuronal functions including excitability, gene expression, and synaptic transmission. Four genes lead to developmental and differential expression of CaMKII isoforms (α, β, γ, δ). We determined mRNA levels of these isoforms in the dorsal root ganglia (DRG) of adult rats with and without nerve injury in order to determine if differential expression of CaMKII isoforms may contribute to functional differences that follow injury. DRG neurons express mRNA for all four isoforms, and the relative abundance of CaMKII isoforms was γ>α>β=δ, based on the CT values. Following ligation of the 5th lumbar (L5) spinal nerve (SNL), the β isoform did not change, but mRNA levels of both the γ and α isoforms were reduced in the directly injured L5 neurons, and the α isoform was reduced in L4 neurons, compared to their contemporary controls. In contrast, expression of the δ isoform mRNA increased in L5 neurons. CaMKII protein decreased following nerve injury in both L4 and L5 populations. Total CaMKII activity measured under saturating Ca(2+)/CaM conditions was decreased in both L4 and L5 populations, while autonomous CaMKII activity determined in the absence of Ca(2+) was selectively reduced in axotomized L5 neurons 21days after injury. Thus, loss of CaMKII signaling in sensory neurons after peripheral nerve injury may contribute to neuronal dysfunction and pain.