2 )], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA 2 by muscarinic stimulation. Third, in type IIa PLA 2 [secreted (sPLA 2 )] ؊/؊ / cPLA 2 ؊/؊ double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M 1 R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AA's concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated CaVβ2a.

During direct membrane depolarization, Ca2+ influx primarily through L-type Ca2+ (L-) channels initiates activity-dependent gene transcription. This is surprising given that in most neurons a minority of the total Ca2+ current arises from L-channel activity. However, many studies have stimulated Ca2+ influx with unphysiological stimuli such as chronic membrane depolarization using high K+ medium. Few studies have tested whether other Ca2+ channels stimulate gene transcription in adult neurons as a consequence of direct electrical stimulation. Therefore, we evaluated the role of L- and N-type Ca2+ (N-) channel activity in regulating mRNA levels of c-fos, an activity-dependent transcription factor, in adult rat superior cervical ganglion (SCG) neurons as the majority of Ca2+ channels are N-type, while only a minority are L-type. Changes in c-fos mRNA levels were measured using semi-quantitative and single-cell RT-PCR. Phosphorylation of CREB (pCREB) and changes in c-Fos levels were visualized in dissociated cells by immunocytochemistry. Increases in pCREB, c-fos mRNA and c-Fos protein with either K+ or electrical depolarization required Ca2+ influx. These results support previous findings that elevated c-fos levels result from pCREB stimulating c-fos transcription. Elevation of pCREB, c-fos and c-Fos with K+ depolarization depended on L-channel activity. By contrast, antagonizing either channel at 10-Hz stimulation minimized these increases despite unequal numbers of the two channel types. Transition to exclusive L-channel involvement occurred with increasing frequency of stimulation (from 10 to 20 to 50 Hz). Our results demonstrate that N- and L-channel participation in regulating c-fos expression is encoded in the pattern of electrical stimulation.

A near infrared calibration model with higher precision and better stability was constructed in the present study, using 280 cottonseed samples. The reference phytic acid contents were determined by high-performance ion chromatography. A combination of Savitzky-Golay smoothing, standard normal variate, and the first derivative was chosen as the spectral pre-treatment method. Monte Carlo uninformative variable elimination was proposed for spectral variable selection. The regression methods of partial least squares, least squares support vector machines, and weighted least squares support vector machines were developed for the calibration model. The optimal near infrared calibration model for phytic acid contents in the cottonseed meals was least squares support vector machines, with r 2 ¼ 0.97, RPD ¼ 5.53, RMSECV ¼ 0.06%, and RMSEP ¼ 0.05%. This robust method can replace the traditional method of phytic acid determination in cottonseed meals.

Background: Manganese (Mn) is an essential microelement in cottonseeds, which is usually determined by the techniques relied on hazardous reagents and complex pretreatment procedures. Therefore a rapid, low-cost, and reagent-free analytical way is demanded to substitute the traditional analytical method. Results: The Mn content in cottonseed meal was investigated by near-infrared spectroscopy (NIRS) and chemometrics techniques. Standard normal variate (SNV) combined with first derivatives (FD) was the optimal spectra pre-treatment method. Monte Carlo uninformative variable elimination (MCUVE) and successive projections algorithm method (SPA) were employed to extract the informative variables from the full NIR spectra. The linear and nonlinear calibration models for cottonseed Mn content were developed. Finally, the optimal model for cottonseed Mn content was obtained by MCUVE-SPA-LSSVM, with root mean squares error of prediction (RMSEP) of 1.994 6, coefficient of determination (R 2 ) of 0.949 3, and the residual predictive deviation (RPD) of 4.370 5, respectively. Conclusions: The MCUVE-SPA-LSSVM model is accuracy enough to measure the Mn content in cottonseed meal, which can be used as an alternative way to substitute for traditional analytical method.