Calcium Channel Protein Expression Research Articles

An efficient nonlinear hybridization chain reaction-based sensitive fluorescent assay for in situ estimation of calcium channel protein expression on bone marrow cells

A sensitive and highly efficient approach to monitor the expression of proteins on live cells was urgently needed to demonstrate its factor and mechanism and most important for clinical diagnostics and molecular biology. Herein, we developed a simple and highly efficient strategy, nonlinear hybridization chain reaction (nonlinear HCR), for the sensitive determination of proteins on live cells with transient receptor potential vanilloid 4 (TRPV4) and RAW264.7 cells as a model. Unlike the normal hybridization chain reaction (HCR) with multiplicative amplification, an exponential amplified fluorescent response could be obtained in theory based on the proposed nonlinear HCR. As a result, the nonlinear HCR generated a significant enhancement about 3 times compared with the normal HCR and 10 times compared with the directly immunofluorescence assay. Based on the proposed nonlinear HCR, the fluorescent signals increased with the concentration of TRPV4 in the range from 10 pg/mL to 100 ng/mL with a detection limit of 2.8 pg/mL, which would be useful for the sensitive detection of proteins in cell lysis or on cell surface. At the same time, the significant improvements via nonlinear HCR were achieved in the fluorescent imaging system compared with traditional immunofluorescence staining and normal HCR, proving the significant value of nonlinear HCR-based amplification strategy. Success in the establishment of the highly efficient nonlinear HCR strategy offered a simple and sensitive approach to demonstrate the concentration of special proteins on cell and other proteins and nucleotide potentially, revealing a simple and efficient technology for research fields of clinical diagnostics and molecular biology.

Long non-coding RNA CACNA1G-AS1 promotes calcium channel protein expression and positively affects human keloid fibroblast migration.

Keloids are a type of benign hyperplasia that cause dermatologic dysfunction and esthetic deformity by invading adjacent normal tissues. Little is known about their etiology, therefore, they are a challenge to treat using plastic surgery. In a previous study, it was demonstrated that the expression of the long non-coding RNA CACNA1G-AS1 (CAS1) is high in keloid tissue, suggesting that CAS1 is involved in keloid formation. In the present study, the aim was to identify potential keloid target proteins by exploring CAS1 biological function during cell proliferation and migration, cytokine secretion, collagen secretion and the control of calcium channel protein expression in human keloid fibroblasts. Three biopsy samples were collected from each patient with keloids at The Peking Union Medical College Hospital, which were then used to investigate the role of CAS1 in cell proliferation and migration. CAS1 silencing was also carried out using small interfering RNA; cell factors, collagen and calcium channel protein levels were compared with control cells. The interference of CAS1 expression reached 50% compared with the control group. CACNA1G and type I collagen expression was significantly downregulated by CAS1 knockdown, while the expression of transforming growth factor-β and type III collagen was not affected. Wound healing time was longer in the CAS1-knockdown group, but there was no visible change in cell proliferation. In conclusion, CAS1 appeared to promote calcium channel protein and type I collagen expression, and to have a positive effect on cell migration in human keloid fibroblasts. Therefore it has potential as a novel therapeutic target for keloids.

S-adenosyl methionine regulates calcium channels and inhibits uterine smooth muscle contraction in rats with infectious premature delivery through the transient receptor protein 3/protein kinase Cβ/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa signaling pathway.

The aim of the present study was to investigate the effects of S-adenosyl methionine (SAMe) on infectious premature inflammatory factors and uterine contraction, and to further explore its mechanism of action via the transient receptor protein 3 (TRPC3)/protein kinase Cβ (PKCβ)/C-kinase-activated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) signaling pathway, following intervention by a TRPC3 inhibitor. A rat model of premature delivery induced by lipopolysaccharide (LPS) was established. Following treatment with SAMe and inhibiting TRPC3 expression, rat serum and uterus were isolated. Hematoxylin and eosin staining was used to observe the histopathological changes in the uterus. Uterine muscle strips in vitro were selected to measure the changes in muscle tension. ELISA was utilized to measure the changes in serum inflammatory factor and oxidative stress indexes. Immunohistochemistry, western blot assay and reverse transcription-quantitative polymerase chain reaction were applied to detect calcium channel protein expression in the uterus. Western blot analysis was employed to measure the expression of TRPC3/PKCβ/CPI-17 signaling pathway-related proteins. TRPC3 was highly expressed in the uterus of rat models of premature delivery induced by LPS. Following treatment with SAMe, inflammatory cell infiltration markedly reduced in the uterus and the tension of in vitro uterine muscle strips significantly decreased. SAMe treatment suppressed inflammatory reaction and oxidative stress, and diminished L-type and T-type calcium channel protein expression. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression was also reduced. When TRPC3 expression was suppressed, the effects of SAMe against inflammation and oxidative stress were diminished. TRPC3/PKCβ/CPI-17 signaling pathway-related protein expression significantly increased. SAMe was able to reduce inflammatory reaction and oxidative stress in the uterus of rat model of infectious premature delivery induced by LPS, prolong delivery time, reduce the mortality rate of offspring rats, and serve a therapeutic role. This effect is likely achieved via the regulation of uterine contractions and childbirth through the TRPC3/PKCβ/CPI-17 signaling pathway.

Effects of calcium channel on ovarian cancer cells.

The aim of the present study was to examine the effects of calcium channel protein on ovarian cancer cells. The expression of calcium channel protein in normal ovarian cells and ovarian cancer cells was detected by fluorescence quantitative PCR. Subsequently, the ovarian cancer cells were added to calcium channel protein activator media at various concentrations of 0, 1, 4, 8, 12, 16 and 20 mmol/l. The concentration of calcium ion in different samples was produced, and using an MTT assay, ovarian cancer cell activity in various samples was detected. Finally, a flow cytometer was used to explore the apoptosis rate. It was found that there was a significant difference between the expression of calcium channel protein in normal ovarian tissue and ovarian cancer cells (P<0.05), as well as a significant difference of calcium concentration among various samples (P<0.05). When the concentrations of calcium channel activator were 1, 4, 8, 12, 16 and 20 mmol/l, the values of the ovarian cancer cell inhibition rates were 4.6, 21.3, 48.3, 67.9, 52.8 and 31.8%, respectively. It showed that the calcium channel activator inhibited the proliferation of ovarian cancer cells to a certain extent, in a dose-dependent manner, especially when the concentration was at 12 mmol/l at which the intracellular calcium concentration was similar to that in normal ovarian cells. In conclusion, calcium ions play an important role in promoting cell proliferation of ovarian cancer cells, and they were involved in apoptosis of ovarian cancer cells to some extent, which regulates apoptosis by controlling the content of intracellular calcium.

Selective Loss of Smaller Spines in Schizophrenia.

Decreased density of dendritic spines in adult schizophrenia subjects has been hypothesized to result from increased pruning of excess synapses in adolescence. In vivo imaging studies have confirmed that synaptic pruning is largely driven by the loss of large or mature synapses. Thus, increased pruning throughout adolescence would likely result in a deficit of large spines in adulthood. Here, the authors examined the density and volume of dendritic spines in deep layer 3 of the auditory cortex of 20 schizophrenia and 20 matched comparison subjects as well as aberrant voltage-gated calcium channel subunit protein expression linked to spine loss. Primary auditory cortex deep layer 3 spine density and volume was assessed in 20 pairs of schizophrenia and matched comparison subjects in an initial and replication cohort (12 and eight pairs) by immunohistochemistry-confocal microscopy. Targeted mass spectrometry was used to quantify postsynaptic density and voltage-gated calcium channel protein expression. The effect of increased voltage-gated calcium channel subunit protein expression on spine density and volume was assessed in primary rat neuronal culture. Only the smallest spines are lost in deep layer 3 of the primary auditory cortex in subjects with schizophrenia, while larger spines are retained. Levels of the tryptic peptide ALFDFLK, found in the schizophrenia risk gene CACNB4, are inversely correlated with the density of smaller, but not larger, spines in schizophrenia subjects. Consistent with this observation, CACNB4 overexpression resulted in a lower density of smaller spines in primary neuronal cultures. These findings require a rethinking of the overpruning hypothesis, demonstrate a link between small spine loss and a schizophrenia risk gene, and should spur more in-depth investigations of the mechanisms that govern new or small spine generation and stabilization under normal conditions as well as how this process is impaired in schizophrenia.

Oxidized Low-density Lipoprotein (ox-LDL) Cholesterol Induces the Expression of miRNA-223 and L-type Calcium Channel Protein in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common sustained arrhythmia causing high morbidity and mortality. While changing of the cellular calcium homeostasis plays a critical role in AF, the L-type calcium channel α1c protein has suggested as an important regulator of reentrant spiral dynamics and is a major component of AF-related electrical remodeling. Our computational modeling predicted that miRNA-223 may regulate the CACNA1C gene which encodes the cardiac L-type calcium channel α1c subunit. We found that oxidized low-density lipoprotein (ox-LDL) cholesterol significantly up-regulates both the expression of miRNA-223 and L-type calcium channel protein. In contrast, knockdown of miRNA-223 reduced L-type calcium channel protein expression, while genetic knockdown of endogenous miRNA-223 dampened AF vulnerability. Transfection of miRNA-223 by adenovirus-mediated expression enhanced L-type calcium currents and promoted AF in mice while co-injection of a CACNA1C-specific miR-mimic counteracted the effect. Taken together, ox-LDL, as a known factor in AF-associated remodeling, positively regulates miRNA-223 transcription and L-type calcium channel protein expression. Our results implicate a new molecular mechanism for AF in which miRNA-223 can be used as an biomarker of AF rheumatic heart disease.

Silver nanoparticle exposure induces rat motor dysfunction through decrease in expression of calcium channel protein in cerebellum

Silver nanoparticles (AgNPs) are currently used widely, however, their impact on central nervous system still remains ambiguous and needs to be elucidated. This study is performed to investigate the neurotoxicity of AgNPs and illustrate the potential molecular mechanism. Neonatal Sprague–Dawley (SD) rats are exposed to AgNPs by intranasal instillation for 14 weeks. It is demonstrated that AgNPs exposure causes cerebellar ataxia like symptom in rats, evidenced by dysfunction of motor coordination and impairment of locomotor activity. Observation of cerebellum section reveals that AgNPs can provoke destruction of cerebellum granular layer with concomitant activation of glial cells. AgNPs treatment decreases calcium channel protein (CACNA1A) levels in cerebellum without changing potassium channel protein (KCNA1) levels. The levels of silver in rat cerebellum tissue are correlated with exposure doses. In vitro experiments reveal that AgNPs treatment significantly reduces the protein and mRNA levels of CACNA1A in primary cultured cerebellum granule cells (CGCs). These findings suggest that AgNPs-induced rat motor dysfunction is associated with CACNA1A expression decrease, which reveals the underlying molecular mechanism for the neurotoxicity of AgNPs. Possible counteractions may accordingly be suggested to attenuate the unexpected harmful effects in biological applications of AgNPs.

Presenilin-null cells have altered two-pore calcium channel expression and lysosomal calcium: Implications for lysosomal function

Presenilins are necessary for calcium homeostasis and also for efficient proteolysis through the autophagy/lysosome system. Presenilin regulates both endoplasmic reticulum calcium stores and autophagic proteolysis in a γ-secretase independent fashion. The endo-lysosome system can also act as a calcium store, with calcium efflux channels being recently identified as two-pore channels 1 and 2. Here we investigated lysosomal calcium content and the channels that mediate calcium release from these acidic stores in presenilin knockout cells. We report that presenilin loss leads to a lower total lysosomal calcium store despite the buildup of lysosomes found in these cells. Additionally, we find alterations in two-pore calcium channel protein expression, with loss of presenilin preventing the formation of a high molecular weight species of TPC1 and TPC2. Finally, we find that treatments that disturb lysosomal calcium release lead to a reduction in autophagy function yet lysosomal inhibitors do not alter two-pore calcium channel expression. These data indicate that alterations in lysosomal calcium in the absence of presenilins might be leading to disruptions in autophagy.

Variation of T-type calcium channel protein expression affects cell division of cultured tumor cells

In this study we investigated the T-type calcium channel and its involvement in the cell division of U87MG cultured glioma cells and N1E-115 neuroblastoma cells. Using Western blot analysis, we found that expression of both α1G and α1H subunits of the T-type calcium channel decreased during conditions associated with a decrease in proliferation as evidenced by increased expression of cyclin D1, a marker for non-proliferating cells. Both serum starvation and application of mibefradil, a selective T-type calcium channel antagonist, resulted in a 50% decrease in the expression of α1G and α1H and a 700–900% increase in levels of cyclin D1 in U87MG and N1E-115 cells, respectively. Furthermore, overexpression of the α1H subunit resulted in a two-fold increase in cell proliferation compared to control cultures or cultures receiving an empty vector. In contrast, blocking expression of the α1G subunit using antisense oligonucleotides lead to a 70% decrease in proliferation of U87MG and N1E-115 cells compared to control cultures or cultures receiving a scrambled oligonucleotide. Our findings suggest that proliferation of U87MG glioma cells and N1E-115 is regulated by T-type calcium channel expression.

THE DECOMPENSATED DETRUSOR IV: EXPERIMENTAL BLADDER OUTLET OBSTRUCTION AND ITS FUNCTIONAL CORRELATION TO THE EXPRESSION OF THE RYANODINE AND VOLTAGE OPERATED CALCIUM CHANNELS

Calcium ion homeostasis has a significant role in smooth muscle function. Its regulation requires complex storage and release mechanisms via ion pumps and channels located within intracellular storage sites (sarcoplasmic reticulum) and at the plasma membrane. A prominent component of the sarcoplasmic reticulum is the ryanodine sensitive ion channel which releases calcium from the sarcoplasmic reticulum into the cytosol. At the level of the plasma membrane the voltage operated calcium channel (dihydropyridine sensitive) serves to allow an influx of extracellular calcium. Our prior studies have shown a loss of sarcoplasmic endoplasmic reticulum Ca++Mg++ATPase expression following outlet obstruction. In this study we correlate ryanodine and voltage operated calcium channel protein expression with bladder function following partial outlet obstruction. Standardized partial bladder outlet obstructions were created in adult New Zealand white rabbits, which were divided into control, sham operated and obstructed groups. Muscle strip studies further subcategorized the obstructed group into compensated (force greater than 50% of control) and decompensated (force less than 50% of control) and were correlated with in vivo determinations of voiding frequency and voided volumes. Microsomal membrane protein fractions were prepared from the same bladder tissue and were used for Western blot analysis using specific monoclonal antibodies. Increased voiding frequency and decreased voided volumes correlated with the definitions of compensated and decompensated. The Western blots revealed a near disappearance of ryanodine expression in the decompensated group with minimal changes in the expression of the voltage operated calcium channel. Bladder performance as measured in vivo and in vitro after outlet obstruction is influenced in part by smooth muscle cell ability to maintain calcium homeostasis via the sarcoplasmic reticulum. Bladder decompensation is highly associated with a loss of sarcoplasmic reticulum function with lesser changes taking place in those calcium regulatory proteins at the plasma membrane.

The decompensated detrusor IV: experimental bladder outlet obstruction and its functional correlation to the expression of the ryanodine and voltage operated calcium channels.

Calcium ion homeostasis has a significant role in smooth muscle function. Its regulation requires complex storage and release mechanisms via ion pumps and channels located within intracellular storage sites (sarcoplasmic reticulum) and at the plasma membrane. A prominent component of the sarcoplasmic reticulum is the ryanodine sensitive ion channel which releases calcium from the sarcoplasmic reticulum into the cytosol. At the level of the plasma membrane the voltage operated calcium channel (dihydropyridine sensitive) serves to allow an influx of extracellular calcium. Our prior studies have shown a loss of sarcoplasmic endoplasmic reticulum Ca++Mg++ATPase expression following outlet obstruction. In this study we correlate ryanodine and voltage operated calcium channel protein expression with bladder function following partial outlet obstruction. Standardized partial bladder outlet obstructions were created in adult New Zealand white rabbits, which were divided into control, sham operated and obstructed groups. Muscle strip studies further subcategorized the obstructed group into compensated (force greater than 50% of control) and decompensated (force less than 50% of control) and were correlated with in vivo determinations of voiding frequency and voided volumes. Microsomal membrane protein fractions were prepared from the same bladder tissue and were used for Western blot analysis using specific monoclonal antibodies. Increased voiding frequency and decreased voided volumes correlated with the definitions of compensated and decompensated. The Western blots revealed a near disappearance of ryanodine expression in the decompensated group with minimal changes in the expression of the voltage operated calcium channel. Bladder performance as measured in vivo and in vitro after outlet obstruction is influenced in part by smooth muscle cell ability to maintain calcium homeostasis via the sarcoplasmic reticulum. Bladder decompensation is highly associated with a loss of sarcoplasmic reticulum function with lesser changes taking place in those calcium regulatory proteins at the plasma membrane.