Changes in the expression of satellite glial cell-specific markers during postnatal development of rat sympathetic ganglia.

The sympathetic ganglia represent a final motor pathway that mediates homeostatic "fight and flight" responses in the visceral organs. Satellite glial cells (SGCs) form a thin envelope close to the neuronal cell body and synapses in the sympathetic ganglia. This unique morphological feature suggests that neurons and SGCs form functional units for regulation of sympathetic output. In the present study, we addressed whether SGC-specific markers undergo age-dependent changes in the postnatal development of rat sympathetic ganglia. We found that fatty acid-binding protein 7 (FABP7) is an early SGC marker, whereas the S100B calcium-binding protein, inwardly rectifying potassium channel, Kir4.1 and small conductance calcium-activated potassium channel, SK3 are late SGC markers in the postnatal development of sympathetic ganglia. Unlike in sensory ganglia, FABP7 + SGC was barely detectable in adult sympathetic ganglia. The expression of connexin 43, a gap junction channel gradually increased with age, although it was detected in both SGCs and neurons in sympathetic ganglia. Glutamine synthetase was expressed in sensory, but not sympathetic SGCs. Unexpectedly, the sympathetic SGCs expressed a water-selective channel, aquaporin 1 instead of aquaporin 4, a pan-glial marker. However, aquaporin 1 was not detected in the SGCs encircling large neurons. Nerve injury and inflammation induced the upregulation of glial fibrillary acidic protein, suggesting that this protein is a hall marker of glial activation in the sympathetic ganglia. In conclusion, our findings provide basic information on the in vivo profiles of specific markers for identifying sympathetic SGCs at different stages of postnatal development in both healthy and diseased states.

Store-operated calcium entry in the satellite glial cells of rat sympathetic ganglia.

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca2+ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca2+ entry (SOCE) which is critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca2+ stores were depleted in the absence of extracellular Ca2+, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca2+ entry channels. Intracellular Ca2+ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca2+ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

Functional plasticity of cardiac efferent neurons contributes to traumatic brain injury-induced cardiac autonomic dysfunction.

Traumatic brain injury (TBI) frequently causes cardiac autonomic dysfunction (CAD), irrespective of its severity, which is associated with an increased morbidity and mortality in patients. Despite the significance of probing the cellular mechanism underlying TBI-induced CAD, animal studies on this mechanism are lacking. In the current study, we tested whether TBI-induced CAD is associated with functional plasticity in cardiac efferent neurons. In this regard, TBI was induced by a controlled cortical impact in rats. Assessment of heart rate variability and baroreflex sensitivity indicated that CAD was developed in the sub-acute period after moderate and severe TBI. The cell excitability was increased in the stellate ganglion (SG) neurons and decreased in the intracardiac ganglion (ICG) neurons in TBI rats, compared with the sham-operated rats. The transient A-type K+ (KA) currents, but not the delayed rectifying K+ currents were significantly decreased in SG neurons in TBI rats, compared with sham-operated rats. Consistent with these electrophysiological data, the transcripts encoding the Kv4 α subunits were significantly downregulated in SG neurons in TBI rats, compared with sham-operated rats. TBI causes downregulation and upregulation of M-type K+ (KM) currents and the KCNQ2 mRNA transcripts, which may contribute to the hyperexcitability of the SG neurons and the hypoexcitability of the ICG neurons, respectively. In conclusion, the key cellular mechanism underlying the TBI-induced CAD may be the functional plasticity of the cardiac efferent neurons, which is caused by the regulation of the KA and/or KM currents.

Increased expression of neuregulin 1 in the urothelium of rat bladder with partial bladder outlet obstruction.

BACKGROUND: This study determined whether changes in the expression of neuregulin (NRG) 1, erbB2 tyrosine kinase (ErbB2) and the M2 muscarinic receptor in the urothelium and detrusor muscle of the rat bladder were associated with partial bladder outlet obstruction (PBOO). METHODS: Male Sprague-Dawley rats (body weight 250-300 g) were used and subdivided into control (n = 10) and PBOO groups (n = 20). PBOO was induced for 21 days, and the expression of NRG1, ErbB2 and M2 muscarinic receptor mRNA and protein was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting, respectively. RESULTS: In the urothelium of rat bladder samples, protein expression and mRNA expression of NRG1, ErbB2 and M2 muscarinic receptor were significantly increased in the PBOO group compared to the control group (p < 0.05). Only mRNA expression levels of NRG1/ ErbB2 were higher in the detrusor muscle of the PBOO group compared to the control group (p < 0.05). CONCLUSIONS: Our study demonstrated remarkable changes in the expression of NRG1/ErbB2 receptor mRNA and protein in the urothelium and muscle layer. These results suggest that NRG1 overexpression plays some kind of role against the PBOO-induced upregulated muscarinic receptors in detrusor overactivity.

Decreased excitability and voltage-gated sodium currents in aortic baroreceptor neurons contribute to the impairment of arterial baroreflex in cirrhotic rats.

Cardiovascular autonomic dysfunction, which is manifested by an impairment of the arterial baroreflex, is prevalent irrespective of etiology and contributes to the increased morbidity and mortality in cirrhotic patients. However, the cellular mechanisms that underlie the cirrhosis-impaired arterial baroreflex remain unknown. In the present study, we examined whether the cirrhosis-impaired arterial baroreflex is attributable to the dysfunction of aortic baroreceptor (AB) neurons. Biliary and nonbiliary cirrhotic rats were generated via common bile duct ligation (CBDL) and intraperitoneal injections of thioacetamide (TAA), respectively. Histological and molecular biological examinations confirmed the development of fibrosis in the livers of both cirrhotic rat models. The heart rate changes during phenylephrine-induced baroreceptor activation indicated that baroreflex sensitivity was blunted in the CBDL and TAA rats. Under the current-clamp mode of the patch-clamp technique, cell excitability was recorded in DiI-labeled AB neurons. The number of action potential discharges in the A- and C-type AB neurons was significantly decreased because of the increased rheobase and threshold potential in the CBDL and TAA rats compared with sham-operated rats. Real-time PCR and Western blotting indicated that the NaV1.7, NaV1.8, and NaV1.9 transcripts and proteins were significantly downregulated in the nodose ganglion neurons from the CBDL and TAA rats compared with the sham-operated rats. Consistent with these molecular data, the tetrodotoxin-sensitive NaV currents and the tetrodotoxin-resistant NaV currents were significantly decreased in A- and C-type AB neurons, respectively, from the CBDL and TAA rats compared with the sham-operated rats. Taken together, these findings implicate a key cellular mechanism in the cirrhosis-impaired arterial baroreflex.

Neuregulin 1 as an endogenous regulator of nicotinic acetylcholine receptors in adult major pelvic ganglion neurons.

We investigated whether endogenous neuregulin 1 (NRG1) is released in a soluble form (called sNRG1) and upregulates expression of nicotinic acetylcholine receptor (nAChR) in autonomic major pelvic ganglion (MPG) neurons of adult rats. To elicit the release of sNRG1, either the hypogastric nerve or the pelvic nerve was electrically stimulated. Then, the MPG-conditioned medium (CM) was subjected to western blotting using an antibody directed against the N-terminal ectodomain of NRG1. Both sympathetic and parasympathetic nerve activation elicited the release of sNRG1 from MPG neurons in a frequency-dependent manner. The sNRG1 release was also induced by treatment of MPG neurons with either high KCl or neurotrophic factors. The biological activity of the released sNRG1 was detected by tyrosine phosphorylation (p185) of the ErbB2 receptors in MPG neurons. When MPG neurons were incubated for 6 h in the CM, the protein level of the nAChR α3 subunit and ACh-induced current (IACh) density were significantly increased. The CM-induced changes in IACh was abolished by a selective ErbB2 tyrosine kinase inhibitor. Taken together, these data suggest that NRG1 functions as an endogenous regulator of nAChR expression in adult MPG neurons.

Bladder outlet obstruction causes up-regulation of nicotinic acetylcholine receptors in bladder-projecting pelvic ganglion neurons.

Pelvic ganglion (PG) neurons relay sympathetic and parasympathetic signals to the lower urinary tract, comprising the urinary bladder and bladder outlet, and are thus essential for both storage and voiding reflexes. Autonomic transmission is mediated by activation of the nicotinic acetylcholine receptor (nAChR) in PG neurons. Previously, bladder outlet obstruction (BOO), secondary to benign prostatic hyperplasia, was found to increase soma sizes of bladder-projecting PG neurons. To date, however, it remains unknown whether these morphological changes are accompanied by functional plasticity in PG neurons. In the present study, we investigated whether BOO alters acetylcholine receptor (nAChR) transcript expression and current density in bladder PG neurons. Partial ligation of the rat urethra for six weeks induced detrusor overactivity (DO), as observed during cystometrical measurement. In rats exhibiting DO, membrane capacitance of parasympathetic bladder PG neurons was selectively increased. Real-time PCR analysis revealed that BOO enhanced the expression of the transcripts encoding the nAChR α3 and ß4 subunits in PG neurons. Notably, BOO significantly increased ACh-evoked current density in parasympathetic bladder PG neurons, whereas no changes were observed in sympathetic bladder and parasympathetic penile PG neurons. In addition, other ligand-gated ionic currents were immune to BOO in bladder PG neurons. Taken together, these data suggest that BOO causes upregulation of nAChR in parasympathetic bladder PG neurons, which in turn may potentiate ganglionic transmission and contribute to the development of DO.

Neuregulin 1 up-regulates the expression of nicotinic acetylcholine receptors through the ErbB2/ErbB3-PI3K-MAPK signaling cascade in adult autonomic ganglion neurons.

We investigated effects of Neuregulin 1 (NRG1) on the expression of nicotinic acetylcholine receptor (nAChR) in major pelvic ganglion (MPG) from adult rat. MPG neurons were found to express transcripts for type I and III NRG1s as well as α and ß-type epidermal growth factor (EGF)-like domains. Of the four ErbB receptor isoforms, ErbB1, ErbB2, and ErbB3 were expressed in MPG neurons. Treating MPG with NRG1ß significantly increased the transcript and protein level of the nAChR α3 and ß4 subunits. Consistent with these molecular data, nicotinic currents (I(ACh) ) were significantly up-regulated in NRG1ß-treated sympathetic and parasympathetic MPG neurons. In contrast, the type III NRG1 and the α form of the NRG1 failed to alter the I(ACh) . Inhibition of the ErbB2 tyrosine kinase completely abolished the effects of NRG1ß on the I(ACh) . Stimulation of the ErbB receptors by NRG1ß activated the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). Immunoblot analysis revealed that PI3K-mediated activation of Akt preceded Erk1/2 activation in NRG1ß-treated MPG neurons. Furthermore, specific PI3K inhibitors abrogated the phosphorylation of Erk1/2, while inhibition of MEK did not prevent the phosphorylation of Akt. Taken together, these findings suggest that NRG1 up-regulates nAChR expression via the ErbB2/ErbB3-PI3K-MAPK signaling cascade and may be involved in maintaining the ACh-mediated synaptic transmission in adult autonomic ganglia.

Co-localization of activating transcription factor 3 and phosphorylated c-Jun in axotomized facial motoneurons.

Activating transcription factor 3 (ATF3) and c-Jun play key roles in either cell death or cell survival, depending on the cellular background. To evaluate the functional significance of ATF3/c-Jun in the peripheral nervous system, we examined neuronal cell death, activation of ATF3/c-Jun, and microglial responses in facial motor nuclei up to 24 weeks after an extracranial facial nerve axotomy in adult rats. Following the axotomy, neuronal survival rate was progressively but significantly reduced to 79.1% at 16 weeks post-lesion (wpl) and to 65.2% at 24 wpl. ATF3 and phosphorylated c-Jun (pc-Jun) were detected in the majority of ipsilateral facial motoneurons with normal size and morphology during the early stage of degeneration (1-2 wpl). Thereafter, the number of facial motoneurons decreased gradually, and both ATF3 and pc-Jun were identified in degenerating neurons only. ATF3 and pc-Jun were co-localized in most cases. Additionally, a large number of activated microglia, recognized by OX6 (rat MHC II marker) and ED1 (phagocytic marker), gathered in the ipsilateral facial motor nuclei. Importantly, numerous OX6- and ED1-positive, phagocytic microglia closely surrounded and ingested pc-Jun-positive, degenerating neurons. Taken together, our results indicate that long-lasting co-localization of ATF3 and pc-Jun in axotomized facial motoneurons may be related to degenerative cascades provoked by an extracranial facial nerve axotomy.

Phenotype-specific down-regulation of nicotinic acetylcholine receptors in the pelvic ganglia of castrated rats: implications for neurogenic erectile dysfunction.

Pelvic ganglia (PG) play critical roles in relaying sympathetic and parasympathetic information from the spinal cord to the penile vasculature and, controlling the penile reflex. Animal studies have shown that androgen deprivation by castration causes erectile dysfunction (ED). Until now, however, neural mechanisms underlying castration-induced ED remain unclear. Therefore, we examined whether androgen deprivation down-regulates nicotinic acetylcholine receptors (nAchRs), which mediate fast excitatory synaptic transmission in the PG. Toward this end, neurogenic ED was demonstrated by measuring the intracavernous pressure in castrated rats. Real-time PCR analysis revealed that the transcripts encoding nAchR α3/α5/ß4 subunits were significantly down-regulated in the PG neurons. In addition, down-regulation of the nAchR subunits was reversed by replacement of testosterone. Patch-clamp experiments showed that the nAchR currents were selectively attenuated in the parasympathetic PG neurons innervating the penile vasculature, activation of which elicits penile erection. Taken together, our data suggest that phenotype-specific down-regulation of nAchRs in the PG neurons may contribute to the neurogenic ED in castrated rats.

Switching-on of serotonergic calcium signaling in activated hepatic stellate cells.

AIM: To investigate serotonergic Ca²+ signaling and the expression of 5-hydroxytryptamine (5-HT) receptors, as well as Ca²+ transporting proteins, in hepatic stellate cells (HSCs). METHODS: The intracellular Ca²+ concentration [Ca²+](i) of isolated rat HSCs was measured with a fluorescence microscopic imaging system. Quantitative PCR was performed to determine the transcriptional levels of 5-HT receptors and endoplasmic reticulum (ER) proteins involved in Ca²+ storage and release in cultured rat HSCs. RESULTS: Distinct from quiescent cells, activated HSCs exhibited [Ca²+](i) transients following treatment with 5-HT, which was abolished by U-73122, a phospholipase C inhibitor. Upregulation of 5-HT(2A) and 5-HT(2B) receptors, but not 5-HT3, was prominent during trans-differentiation of HSCs. Pretreatment with ritanserin, a 5-HT2 antagonist, inhibited [Ca²+](i) changes upon application of 5-HT. Expression of type 1 inositol-5'-triphosphate receptor and type 2 sarcoplasmic/endoplasmic reticulum Ca²+ ATPase were also increased during activation of HSCs and serve as the major isotypes for ER Ca²+ storage and release in activated HSCs. Ca²+ binding chaperone proteins of the ER, including calreticulin, calnexin and calsequestrin, were up-regulated following activation of HSCs. CONCLUSION: The appearance of 5-HT-induced [Ca²+](i) response accompanied by upregulation of metabotropic 5-HT2 receptors and Ca²+ transporting/chaperone ER proteins may participate in the activating process of HSCs.

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats.

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 microM to 1mM). ACh increased both intracellular Ca(2+) ([Ca(2+)](i)) and Na(+) concentrations ([Na(+)](i)) in MPG neurons. The recovery of [Na(+)](i) after the removal of ACh was markedly delayed by ouabain (100 microM), an inhibitor of Na(+)/K(+) ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2+/-5.4% (n=7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca(2+)](i) with BAPTA/AM (20 microM) or the blockade of small-conductance Ca(2+)-activated K(+) channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na(+)](i) and [Ca(2+)](i), which activates Na(+)/K(+) ATPase and Ca(2+)-activated K(+) channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.

Characteristics of P2X7-like receptor activated by adenosine triphosphate in HIT-T15 cells.

OBJECTIVES: The study examined the presence of a P2X7 receptor subtype and its functional roles in pancreatic beta cells. METHODS: In a hamster beta-cell line, HIT-T15 cells, purinergic stimulation was investigated using fluorometry, electrophysiology, flow cytometry, and electrophoresis. RESULTS: Adenosine triphosphate (ATP) and 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) increased in the intracellular free Ca2+ concentration, with an EC50 of 398.0 and 136.6 microM, respectively. Preincubation with oxidized ATP, a P2X7 receptor antagonist, inhibited the ATP- and BzATP-induced increase in the intracellular Ca2+ level. The BzATP-induced increase in the intracellular Ca2+ level was dependent on the extracellular Ca2+ concentration. The extracellular Mg2+ had a significant effect on the ATP-induced increase in the intracellular Ca2+ level. The ATP also induced depolarization like high potassium chloride. In the voltage-clamp experiments, ATP evoked inward currents, which were reversed at almost 0 mV. The ATP stimulated the slow influx of ethidium bromide, indicating permeability to larger molecules. Flow cytometry showed that the number of hypodiploid cells (A0), which are indicative of apoptosis, increased when the cells were exposed to ATP for 24 hours. The ATP also induced DNA fragmentation. CONCLUSIONS: These results suggest that the HIT-T15 cells have endogenous P2X7-like receptors and that purinergic stimulation increased the level of intracellular Ca2+, depolarization, inward current, permeability, and apoptosis.

3D pharmacophore based virtual screening of T-type calcium channel blockers.

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.

Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density.

T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Cav3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4alpha-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II-III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.

An alpha3beta4 subunit combination acts as a major functional nicotinic acetylcholine receptor in male rat pelvic ganglion neurons.

We identified major subunits of the nicotinic acetylcholine receptor (nAChR) involved in excitatory postsynaptic potential and intracellular Ca(2+) ([Ca(2+)]i) increase in the major pelvic ganglion (MPG) neurons of the male rat. ACh elicited fast inward currents in both sympathetic and parasympathetic MPG neurons. Mecamylamine, a selective antagonist for alpha3beta4 nAChR, potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons (IC(50); 0.53 and 0.22 microM, respectively). Furthermore, alpha-conotoxin AuIB (10 microM), a new selective antagonist for alpha3beta4 nAChR, blocked more than 80% of the ACh-induced currents in MPG neurons. Conversely, alpha-bungarotoxin, alpha-methyllycaconitine, and dihydro-beta-erythroidine, known as blockers of the alpha7 or alpha4beta2, did not show selective blocking effects on MPG neurons. ACh transiently increased [Ca(2+)]i which was subsequently abolished in the extracellular Ca(2+)-free environment. Simultaneous recording of [Ca(2+)]i and ionic currents revealed that ACh increased [Ca(2+)]i under the conditions of the voltage-clamped (at -80 mV) state, and this resulted from the influx through nAChR itself. ACh-induced [Ca(2+)]i increase was blocked by mecamylamine (10 microM), but was not affected by atropine (1 microM). RT-PCR analysis showed that, among subunits of nAChR, alpha3 and beta4 were predominantly expressed in MPG. We suggest that activation of alpha3 and beta4 nAChR subunits in MPG neurons induce fast inward currents and [Ca(2+)]i increase, possibly mediating a major role in pelvic autonomic synaptic transmission.

Expression of GABAA receptor beta2/3 subunits in the rat major pelvic ganglion.

Several pharmacological and physiological studies have suggested that GABA(A) receptors (GABA(A) Rs) may exist in the rat major pelvic ganglion (MPG), a large coalescent pelvic ganglion that contains both sympathetic and parasympathetic components which innervates pelvic organs. However, the presence of GABA(A) R in the MPG has never been demonstrated directly by morphological studies. In the present study, we used immunohistochemistry to demonstrate the existence of GABA(A) R beta2/3 subunits for the first time in the rat MPG. We also analyzed the neurochemical properties of MPG neurons expressing GABA(A) R beta2/3 subunits. GABA(A) R beta2/3-immunoreactive (-IR) neurons occupied 27.4+/-7.0% of the whole neuronal population, and many of these (77.6%) were co-localized with tyrosine hydroxylase (TH). Likewise, most (86.5%) of TH-IR neurons were GABA(A) R beta2/3-positive. GABA(A) R beta2/3 subunits were also expressed in a few VIP- or NOS-IR neurons, the cholinergic or non-adrenergic, non-cholinergic (NANC) neurons. These results suggest that GABA(A) Rs are involved in the modulation of most sympathetic, noradrenergic neurons and also a subset of VIP and NOS neurons of the rat MPG.

Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A.

Ca2+ influx through T-type Ca2+ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Cav3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 microM forskolin, an adenylyl cyclase stimulant, increased Cav3.2 channel activity by 40+/-4% over 30 min and negatively shifted the steady-state inactivation curve (V50=-61.4+/-0.2 versus -65.5+/-0.1 mV). Forskolin did not affect other biophysical properties of Cav3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 microM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Cav3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 microM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Cav3.2 channel activity by PKA was mimicked by serotonin when 5HT7 receptor was coexpressed with Cav3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Cav3.2 with those of the Nav1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Cav3.2.

Expression profiles of high voltage-activated calcium channels in sympathetic and parasympathetic pelvic ganglion neurons innervating the urogenital system.

Among the autonomic ganglia, major pelvic ganglia (MPG) innervating the urogenital system are unique because both sympathetic and parasympathetic neurons are colocalized within one ganglion capsule. Sympathetic MPG neurons are discriminated from parasympathetic ones by expression of low voltage-activated Ca2+ channels that primarily arise from T-type alpha1H isoform and contribute to the generation of low-threshold spikes. Until now, however, expression profiles of high voltage-activated (HVA) Ca2+ channels in these two populations of MPG neurons remain unknown. Thus, in the present study, we dissected out HVA Ca2+ channels using pharmacological and molecular biological tools. Reverse transcription-polymerase chain reaction analysis showed that MPG neurons contained transcripts encoding all of the known HVA Ca2+ channel isoforms (alpha1B, alpha1C, alpha1D and alpha1E), with the exception of alpha1A. Western blot analysis and pharmacology with omega-agatoxin IVA (1 microM) confirmed that MPG neurons lack the alpha1A Ca2+ channels. Unexpectedly, the expression profile of HVA Ca2+ channel isoforms was identical in the sympathetic and parasympathetic neurons of the MPG. Of the total Ca2+ currents, omega-conotoxin GVIA-sensitive N-type (alpha1B) currents constituted 57 +/- 5% (n = 9) and 60 +/- 3% (n = 6), respectively; nimodipine-sensitive L-type (alpha1C and alpha1D) currents made up 17 +/- 4% and 14 +/- 2%, respectively; and nimodipine-resistant and omega-conotoxin GVIA-resistant R-type currents were 25 +/- 3% and 22 +/- 2%, respectively. The R-type Ca2+ currents were sensitive to NiCl2 (IC50 = 22 +/- 0.1 microM) but not to SNX-482, which was able to potently (IC50 = 76 +/- 0.4 nM) block the recombinant alpha1E/beta2a/alpha2delta Ca2+ currents expressed in human embryonic kidney 293 cells. Taken together, our data suggest that sympathetic and parasympathetic MPG neurons share a similar but unique profile of HVA Ca2+ channel isoforms.

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels.

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.