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Abstract: The effects of the metal loading (30–70wt.%), metal molar ratio (Co/Fe, 1–5) and mass ratio of citric acid to the catalyst (0–0.6) on the productivity and mean diameter of the multi-walled carbon nanotubes (MWCNTs) in a gas–solid fluidized bed reactor (with an inner diameter of 0.056m and a height of 1.0m) were determined. Liquefied petroleum gas (LPG) was used as the carbon source. X-ray diffraction (XRD) was used to characterize the catalysts synthesized using a combustion method. MWCNTs synthesized in the fluidized bed reactor were characterized by field emission scanning electron microscopy (FE-SEM) to observe their morphologies and measure their diameters. Productivity was increased by increasing both the metal loading and the mass ratio of citric acid to the catalyst. A high productivity, up to 2000%, was obtained. The catalyst transition metal particle grain size decreased in the range of 8–17nm with an increasing citric acid mass ratio to the catalyst and the mean diameter of the MWCNTs decreased with increasing the metal molar ratio, however the correlation between the grain size in the catalyst and the mean diameter of MWCNTs remains unclear. [Copyright &y& Elsevier]

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 4α-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. [ABSTRACT FROM AUTHOR]

Although T-type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Cav3.2 T-type channels reconstituted in Xenopus oocytes. Phorbol-12-myristate-13-acetate (PMA) strongly enhanced the amplitude of Cav3.2 channel currents (∼3-fold). The augmentation effects were not mimicked by 4α-PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Cav3.2 channel activity via activation of oocyte PKC. [Copyright &y& Elsevier]

Abstract: 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 (10μM to 1mM). ACh increased both intracellular Ca2+ ([Ca2+]i) and Na+ concentrations ([Na+]i) in MPG neurons. The recovery of [Na+]i after the removal of ACh was markedly delayed by ouabain (100μM), 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 [Ca2+]i with BAPTA/AM (20μM) or the blockade of small-conductance Ca2+-activated K+ channels by apamin (500nM). Taken together, the activation of nAChR increases [Na+]i and [Ca2+]i, which activates Na+/K+ ATPase and Ca2+-activated K+ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia. [ABSTRACT FROM AUTHOR]

Abstract: Several pharmacological and physiological studies have suggested that GABAA receptors (GABAA 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 GABAA R in the MPG has never been demonstrated directly by morphological studies. In the present study, we used immunohistochemistry to demonstrate the existence of GABAA R β2/3 subunits for the first time in the rat MPG. We also analyzed the neurochemical properties of MPG neurons expressing GABAA R β2/3 subunits. GABAA R β2/3-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 GABAA R β2/3-positive. GABAA R β2/3 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 GABAA Rs are involved in the modulation of most sympathetic, noradrenergic neurons and also a subset of VIP and NOS neurons of the rat MPG. [Copyright &y& Elsevier]

Although nerve injury is known to up- and down-regulate some metabotropic receptors in vagal afferent neurons of the nodose ganglia (NG), the functional significance has not been elucidated. In the present study, thus, we examined whether nerve injury affected receptor-mediated Ca2+ channel modulation in the NG neurons. In this regard, unilateral vagotomy was performed using male Sprague–Dawley rats. One week after vagotomy, Ca2+ currents were recorded using the whole-cell variant of patch-clamp technique in enzymatically dissociated NG neurons. In sham controls, norepinephrine (NE)-induced Ca2+ current inhibition was negligible. Following vagotomy, however, the NE responses were dramatically increased. This phenomenon was in accordance with up-regulation of α2A/B-adrenergic receptor mRNAs as quantified using real-time RT-PCR analysis. In addition, neuropeptide Y (NPY) and prostaglandin E2 responses were moderately augmented in vagotomized NG neurons. The altered NPY response appears to be caused by up-regulation of Y2 receptors negatively coupled to Ca2+ channels. In contrast, nerve injury significantly suppressed opioid (tested with DAMGO)-induced Ca2+ current inhibition with down-regulation of μ-receptors. Taken together, these results demonstrated for the first time that the profile of neurotransmitter-induced Ca2+ channel modulation is significantly altered in the NG neurons under pathophysiological state of nerve injury. [Copyright &y& Elsevier]

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., (Copyright © 2016 the American Physiological Society.)

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.

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.