Spinal cord regeneration induced by a voltage-gated calcium channel agonist

Abstract

Regeneration in the central nervous system (CNS) is prohibitive. This is likely due to an interplay of cellular (gene expression, growth factors) and environmental (inhibition by CNS myelin) factors. Calcium supports various intracellular functions, and multiple in vitro studies have shown a role of calcium in axonal growth. In this study, we examine the role of a calcium agonist, S(-)-Bay K 8644, in promoting or impeding CNS growth in vivo, in an effort to understand further the relationship between the voltage-gated L type calcium channel and regeneration. Using a well-established rat spinal cord model of regeneration, we have injected various doses of S(-)-Bay K 8644 (30-240 M) around the injured spinal cord. Our results demonstrate that S(-)-Bay K 8644 enhances regeneration in a dose-dependent fashion. In addition, at very specific concentrations, the same agonist has no effect on or even inhibits regeneration. We conclude that spinal regeneration is highly dependent on intracellular calcium concentration. Furthermore, depending on the dose used, the effect of calcium agonist supplementation on spinal regeneration can be supportive or inhibitory.     

以压迫阻滞针效定位的方法测试人体体表心包经的循行路线

以心功能和心电图为指标,以机械压迫阻滞针效为手段,比较压迫心包经穴位、经线上非穴位点和该穴点两侧对照点对针刺内关穴针效的影响,检测心包经在体表的循行轨迹。结果,压迫经上穴点针后各项指标变化微小,针效显著降低;压迫两侧对照点则对针效无明显影响。两者比较,差异非常显著。证明上肢屈侧确有一条与心包经一致的机能路线,并与心脏机能活动调节密切相关。     

Molecular Basis of Altered Contractility in Experimental Colitis Expression of L-Type Calcium Channel

Colitis in experimental animals or idiopathicinflammatory bowel disease, such as ulcerative colitisor Crohn's disease in humans, is associated with reducedmuscle contraction. This is predicted to be due to disturbance of Ca²⁺homeostasis in the inflamed muscle cell. However, theunderlying molecular mechanism remains to be elucidated.Since the catalytic -1 subunit of the L-typeCa²⁺ channel regulates Ca²⁺ influx, levels of the -1mRNA and protein were examined. Colitis induced byintrarectal administration of trinitrobenzenesulfonicacid was monitored by measuring the myeloperoxidaseactivity and histology. The levels of mRNA and protein wereestimated using RT-PCR and immunoblotting.Myeloperoxidase activity increased in the inflamedcolon, and the lamina propria and muscle layers showedinfiltration of inflammatory cells and loss of crypts. Twoalternatively spliced -1 mRNA isoforms weredetected in the colonic muscle. The ratio of unsplicedto spliced mRNA isoforms remained unaltered in inflamed muscle. In contrast, the level of correspondingprotein isozymes decreased in the colitic animals. Thuscolitis-induced reduction in the -1 protein mayaccount for the reduced colonic contractility seen in colitis.     

Electric Field Stimulation-Induced Guinea Pig Gallbladder Contractions (Role of Calcium Channels in Acetylcholine Release)

Gallbladder motility is modulated by intrinsiccholinergic neurons. The aims of this study were todetermine: (1) the effect of electric field stimulation(EFS) on guinea pig gallbladder smooth muscle, and (2) the role of calcium channels inmediating neurotransmitter release. Gallbladder musclestrips were studied isometrically in vitro. EFS (1-16Hz, 100 V, 0.5-msec pulse width, 30-sec train duration) was used to activate the intrinsic nerves.Exogenous acetylcholine was also used to directlystimulate the smooth muscle. EFS produced afrequency-dependent contractile response that wascompletely abolished by tetrodotoxin. EFS-induced contractions at 16Hz were suppressed by 84 ± 4% with atropine,whereas hexamethonium had no effect. The L-type calciumchannel blocker, nifedipine, reduced EFS contractions by 51 ± 4%, whereas it reduced contractionsto acetylcholine by only 11 ± 5%. The N-typecalcium channel blocker, omega-conotoxin GVIA, reducedEFS-induced contractions by 22 ± 9%, but did notaffect acetylcholine-induced contractions. EFS-induced contractions of theguinea pig gallbladder are primarily mediated byactivation of postganglionic cholinergic neurons. Theacetylcholine release from these cholinergic neurons is regulated by L- and N-type calcium channels.The inhibitory effect of calcium channel blockers on thegallbladder seen in vivo may be in part related toinhibition of acetylcholine release from the intrinsic cholinergic nerves of thegallbladder.     

Involvement of cyclooxygenase, phospholipase C and MAP kinase pathways in human platelet aggregation mediated by the synergistic interaction of adrenaline and PAF

This study was conducted to examine the mechanism(s) of synergistic interaction of adrenaline and platelet-activating factor (PAF) mediated human platelet aggregation. We found that platelet aggregation mediated by subthreshold concentrations of PAF (5-8 nM) plus adrenaline (0.5-2 M) was inhibited by both ₂-adrenoceptor blocker (yohimbine) and PAF receptor antagonist (WEB2086). While examining the role of the downstream signalling pathways, we found that this synergism was inhibited by calcium channel blockers, verapamil, and diltiazem. In addition, platelet aggregation by co-addition of adrenaline and PAF was also inhibited by very low concentrations of phospholipase C (PLC) inhibitor (U73122; IC₅₀ = 0.2 _M), the MAP kinase inhibitor, PD98059 (IC₅₀ = 3 _M) and cyclooxygenase (COX-1) inhibitors including indomethacin (IC₅₀= 0.25 _M), flurbiprofen (IC₅₀ = 0.7 _M) and piroxicam (IC₅₀ = 7 _M). However, the COX-2 inhibitor, nimesulide, was also effective in inhibiting the aggregation. The inhibitors of tyrosine kinase (genistien) and phosphatidylinositol 3-kinase inhibitor (wortmannin) had no significant effects on platelet aggregation. These data suggest that the synergistic effect of adrenaline and PAF on human platelet aggregation is receptor-mediated and involves the activation of PLC/calcium, COX and MAP kinase signalling pathways.     

Calcium/calmodulin-signaling supports TRPV4 activation in osteoclasts and regulates bone mass

Osteoclast differentiation is critically dependent on calcium (Ca(2+)) signaling. Transient receptor potential vanilloid 4 (TRPV4), mediates Ca(2+) influx in the late stage of osteoclast differentiation and thereby regulates Ca(2+) signaling. However, the system-modifying effect of TRPV4 activity remains to be determined. To elucidate the mechanisms underlying TRPV4 activation based on osteoclast differentiation, TRPV4 gain-of-function mutants were generated by the amino acid substitutions R616Q and V620I in TRPV4 and were introduced into osteoclast lineage in Trpv4 null mice to generate Trpv4(R616Q/V620I) transgenic mice. As expected, TRPV4 activation in osteoclasts increased the number of osteoclasts and their resorption activity, thereby resulting in bone loss. During in vitro analysis, Trpv4(R616Q/V620I) osteoclasts showed activated Ca(2+)/calmodulin signaling compared with osteoclasts lacking Trpv4. In addition, studies of Trpv4(R616Q/V620I) mice that lacked the calmodulin-binding domain indicated that bone loss due to TRPV4 activation was abrogated by loss of interactions between Ca(2+)/calmodulin signaling and TRPV4. Finally, modulators of TRPV4 interactions with the calmodulin-binding domain were investigated by proteomic analysis. Interestingly, nonmuscle myosin IIa was identified by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis, which was confirmed by immunoblotting following coimmunoprecipitation with TRPV4. Furthermore, myosin IIa gene silencing significantly reduced TRPV4 activation concomitant with impaired osteoclast maturation. These results indicate that TRPV4 activation reciprocally regulates Ca(2+)/calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function.     

Parathyroid Hormone and the Use of Diuretics and Calcium‐Channel Blockers: The Multi‐Ethnic Study of Atherosclerosis

Thiazide diuretic (TZ) use is associated with higher bone mineral density, whereas loop diuretic (LD) use is associated with lower bone density and incident fracture. Dihydropyridine‐sensitive calcium channels are expressed on parathyroid cells and may play a role in parathyroid hormone (PTH) regulation. The potential for diuretics and calcium‐channel blockers (CCBs) to modulate PTH and calcium homeostasis may represent a mechanism by which they influence skeletal outcomes. We hypothesized that the use of LD and dihydropyridine CCBs is associated with higher PTH, and TZ use is associated with lower PTH. We conducted cross‐sectional analyses of participants treated for hypertension in the Multi‐Ethnic Study of Atherosclerosis who did not have primary hyperparathyroidism or chronic kidney disease (n = 1888). We used adjusted regression models to evaluate the independent association between TZ, LD, and CCB medication classes and PTH. TZ use was associated with lower PTH when compared with non‐TZ use (44.4 versus 46.9 pg/mL, p = 0.02), whereas the use of LD and CCBs was associated with higher PTH when compared with non‐users of each medication class (LD: 60.7 versus 45.5 pg/mL, p < 0.0001; CCB: 49.5 versus. 44.4 pg/mL, p < 0.0001). Adjusted regression models confirmed independent associations between TZ use and lower PTH (β = –3.2 pg/mL, p = 0.0007), and LD or CCB use and higher PTH (LD: β = +12.0 pg/mL, p < 0.0001; CCB: +3.7 pg/mL, p < 0.0001). Among CCB users, the use of dihydropyridines was independently associated with higher PTH (β = +5.0 pg/mL, p < 0.0001), whereas non‐dihydropyridine use was not (β = +0.58 pg/mL, p = 0.68). We conclude that in a large community‐based cohort with normal kidney function, TZ use is associated with lower PTH, whereas LD and dihydropyridine CCB use is associated with higher PTH. These associations may provide a mechanistic explanation linking use of these medications to the development of skeletal outcomes. © 2016 American Society for Bone and Mineral Research.     

Mice Expressing Mutant Trpv4 Recapitulate the Human TRPV4 Disorders

Activating mutations in transient receptor potential vanilloid family member 4 (Trpv4) are known to cause a spectrum of skeletal dysplasias ranging from autosomal dominant brachyolmia to lethal metatropic dysplasia. To develop an animal model of these disorders, we created transgenic mice expressing either wild‐type or mutant TRPV4. Mice transgenic for wild‐type Trpv4 showed no morphological changes at embryonic day 16.5 but did have a delay in bone mineralization. Overexpression of a mutant TRPV4 caused a lethal skeletal dysplasia that phenocopied many abnormalities associated with metatropic dysplasia in humans, including dumbbell‐shaped long bones, a small ribcage, abnormalities in the autopod, and abnormal ossification in the vertebrae. The difference in phenotype between embryos transgenic for wild‐type or mutant Trpv4 demonstrates that an increased amount of wild‐type protein can be tolerated and that an activating mutation of this protein is required to produce a skeletal dysplasia phenotype. © 2014 American Society for Bone and Mineral Research