Association study between polymorphisms in selenoprotein genes and susceptibility to Kashin-Beck disease.

OBJECTIVES: Kashin-Beck disease (KBD) is a disabling osteoarthropathy involving growth cartilage endemic to selenium (Se)-deficient regions in China. Associations between genetic variation in selenoprotein genes and susceptibility to many diseases have recently been investigated but few studies have been performed on KBD. We found four genetic polymorphisms in selenoprotein genes and assessed their association with increased susceptibility to KBD. METHODS: Four polymorphisms including GPX1 (rs1050450), TrxR2 (rs5748469), SEPP1 (rs7579) and DIO2 (rs225014) were analyzed for 161 KBD patients and 312 controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS PCR). Glutathione peroxidase (GPX) activity in whole blood was measured using a GPX assay kit. The mRNA expression of GPX1, nuclear factor-kappaB (NF-kappaB) p65 and p53 in both whole blood and articular cartilage tissue were detected using Real-Time PCR. RESULTS: The genotypic and allelic frequency of GPX1 Pro198Leu was significantly different between KBD patients and controls (P=0.013, P=0.037). A significant increased KBD risk was observed in individuals with Pro/Leu or Leu/Leu (odds ratio=1.781; 95% confidence interval: 1.127-2.814) compared with Pro/Pro. No association was observed between the other three single nucleotide polymorphisms (SNPs) and KBD risk. In addition, GPX enzyme activity in whole blood was lower in the KBD group (P<0.01), and the GPX activity in whole blood decreased significantly in a subgroup of individuals representing Pro/Leu and Leu/Leu compared to Pro/Pro (P<0.01). In whole blood and articular cartilage tissue samples of KBD patients, GPX1 and NF-kappaB p65 mRNA levels were lower (P<0.01) while p53 levels were higher (P<0.001). CONCLUSION: GPX1 Pro198Leu is a potential genetic risk factor in the development of KBD and the GPX1 Leu allele is significantly associated with higher KBD risk among the Chinese Han population and with lower GPX enzyme activity. The expression of apoptosis related molecules in KBD patients significantly differs from controls.

Compensatory recovery of vagal control of hemodynamics after unilateral vagotomy.

This study investigated whether each part of the heart is evenly innervated by the left or right vagus and observed the mechanism of compensatory recovery after unilateral cervical vagotomy. HR, BP, LVSP and +/-dp/dt max all decreased one week after left vagotomy, whereas only BP and -dp/dt max decreased one week after right vagotomy. Western blot analyses revealed that the expression of M(2) receptors in the left atrium and left ventricle was upregulated after subacute (1 week) left/right vagotomy. However, significantly more cholinesterase-positive nerves in LV and RV were seen one week after unilateral vagotomy compared to the sham-operated group. In addition, baroreflex sensitivity was increased after subacute right vagotomy. The decreasing effects of ACh (0.5 microg/kg) on LVSP and +/-dp/dt max (but not on HR and BP) were facilitated by subacute unilateral vagotomy. Our present experiments indicate that 1) the working myocardium is innervated bilaterally by the vagus, 2) ventricular contractility is influenced more by denervation of the left than the right vagus and 3) up-regulation of M(2) muscarinic receptors in the left heart, increase of cholinergic nerves, and high baroreflex sensitivity could be involved in the mechanism of compensatory hemodynamic recovery via contralateral vagus overactivity, thereby amplifying contralateral vagal activity and decreasing cardiac contractility.

Serotonin induces parathyroid hormone-related protein in goat mammary gland.

During lactation, large amounts of calcium are exported from the mammary gland into milk to ensure skeletal growth of the offspring. Recent studies revealed that serotonin (5-HT) is essential to stimulate skeletal calcium resorption for milk synthesis. Our objective was to explore the correlation between circulating 5-HT and serum calcium and parathyroid hormone-related protein (PTHrP) concentrations around parturition in dairy goats. We also investigated the effect of 5-HT on PTHrP expression in cultured primary goat mammary epithelial cells (GMEC). Blood samples of multiparous Guanzhong dairy goats were collected on day -5 to 3 postpartum for analysis of serum concentrations of calcium, 5-HT, and PTHrP. Results revealed that from day -3 to 0 postpartum serum calcium and 5-HT concentrations decreased progressively, but serum PTHrP concentration only had a sharp drop in the postpartum period sampled. Correlation analysis of circulating 5-HT and serum calcium and PTHrP concentrations on day 1 and 2 postpartum revealed that low serum 5-HT concentration was positively correlated with serum total calcium or PTHrP concentration. By knocking down tryptophan hydroxylase-1 (TPH1) or adding 5-hydroxytryptophan (5-HTP) to decrease or increase the levels of 5-HT in GMEC, we observed that 5-HTP increased PTHrP expression in a dose-dependent manner and siTPH1 decreased PTHrP protein expression. Furthermore, 5-HT increased mRNA abundance of calcium-sensing receptor (CaSR) in a dose-dependent manner and decreased the expression of plasma membrane Ca2+ ATPase-1 (PMCA1). Taken together, 5-HT seems to induce PTHrP expression in goat mammary cells during and after parturition. These findings suggest that increasing 5-HT biosynthesis could be a potential therapeutic target for prevention of hypocalcemia in dairy goats.

Identifying differentially expressed genes under heat stress and developing molecular markers in orchardgrass (Dactylis glomerata L.) through transcriptome analysis.

Orchardgrass (Dactylis glomerata L.) is a long-lived, cool-season forage grass that is commonly used for hay production. Despite its economic importance, orchardgrass genome remains relatively unexplored. In this study, we used Illumina RNA sequencing to identify gene-associated molecular markers, including simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), as well as heat stress-induced differentially expressed genes (DEGs) in two orchardgrass genotypes, 'Baoxing' (heat resistant) and '01998' (heat susceptible). Approximately 163 million high-quality trimmed reads were generated from 207 million raw reads using the Illumina HiSeq 2000 platform. A total of 126,846 unigenes were obtained after de novo assembly of the trimmed reads, and 40,078 unigenes were identified as coding sequences (CDSs). Based on the assembled unigenes, 669,300 high-quality SNPs, including 416,099 transitions and 257,736 transversions, were contained in 75,875 unigenes. In addition, a total of 8475 microsatellites were detected in 7764 unigenes. When placed under heat stress, the total number of DEGs in 'Baoxing' (3527) was higher than in '01998' (2649), indicating that in comparison with heat-susceptible '01998', heat-resistant 'Baoxing' seems to have more unigenes that respond to heat stress. The high-throughput transcriptome sequencing of orchardgrass under heat stress provides useful information for gene identification and for the development of SNP and SSR molecular markers. The comparison of DEGs under different periods of heat stress allowed us to identify a wealth of candidate DEGs that can be further analysed in order to determine the genetic mechanisms underlying heat tolerance in orchardgrass.

Graded alpha1-adrenoceptor activation of arteries involves recruitment of smooth muscle cells to produce 'all or none' Ca(2+) signals.

Confocal laser scanning microscopy and Fluo-4 were used to visualize Ca(2+) transients within individual smooth muscle cells (SMC) of rat resistance arteries during alpha(1)-adrenoceptor activation. The typical spatio-temporal pattern of [Ca(2+)] in an artery after exposure to a maximally effective concentration of phenylephrine (PE, 10.0 microM) was a large, brief, relatively homogeneous Ca(2+) transient, followed by Ca(2+) waves, which then declined in frequency over the course of 5 min and which were asynchronous in different SMC. Concentration-Effect (CE) curves relating the concentration of PE (range: 0.1 microM to 10.0 microM) to the effects (fraction of cells producing at least one Ca(2+) wave, and number of Ca(2+) waves during 5 min) had EC(50) values of approximately 0.5 microM and approximately 1.0 microM respectively. The initial Ca(2+) transient and the subsequent Ca(2+) waves were abolished in the presence of caffeine (10.0 mM). A repeated exposure to PE, 1.5 min after the first had ended, elicited fewer Ca(2+) waves in fewer cells than did the initial exposure. Caffeine-sensitive Ca(2+) stores were not depleted at this time, however, as caffeine alone was capable of inducing a large release of Ca(2+)1.5 min after PE. In summary, the mechanism of a graded response to graded alpha(1)-adrenoceptor activation is the progressive 'recruitment' of individual SMC, which then respond in 'all or none' fashion (viz. asynchronous Ca(2+) waves). Ca(2+) signaling continues in the arterial wall throughout the time-course (at least 5 min) of activation of alpha(1)-adrenoceptors. The fact that the Ca(2+) waves are asynchronous accounts for the previously reported fall in 'arterial wall [Ca(2+)]' (i.e. spatial average [Ca(2+)] over all cells).

[Effect of halothane on the muscarinic potassium current of the heart].

Acetylcholine (ACh) released from parasympathetic nerves binds to muscarinic (M2) ACh receptor (mAChR) in the heart, which leads to activation of muscarinic K(+) channel via the betagamma subunit of a G protein. The effect of a general anaesthetic (halothane) on the muscarinic K(+) channel (i(K,Ach)) in guinea-pig atrial cells was investigated using the whole-cell patch clamp technique. Halothane suppressed i(K,Ach), slowed down activation of i(K,Ach) and decreased peak i(K,Ach). When i(K,Ach) was activated by ACh acting via the muscarinic ACh receptors in the normal way, the decrease of i(K,Ach) was greater than the decrease when the muscarinic ACh receptor was bypassed and i(K,Ach) was activated by GTPgammaS. The above finding suggests that the suppression of i(K,Ach) by halothane is, in part, a result of the direct effect on the muscarinic K(+) channel or associated G protein. The decrease of i(K,Ach) by halothane may interfere with parasympathetic control of the heart.

Inhibitory actions of the phosphatidylinositol 3-kinase inhibitor LY294002 on the human Kv1.5 channel.

BACKGROUND AND PURPOSE: Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I(Kur)), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K). EXPERIMENTAL APPROACH: hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method. KEY RESULTS: LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC(50) of 7.9 micromol.L(-1)). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k(+1)) and unbinding (k(-1)) rate constants were calculated to be 1.6 micromol.L(-1) (-1).s(-1) and 5.7 s(-1) respectively. Inhibition by LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A. CONCLUSIONS AND IMPLICATIONS: LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for LY294002.

Comparison of effects of acetylcholine on electromechanical characteristics in guinea-pig atrium and ventricle.

The direct negative effects of acetylcholine (ACh) on guinea-pig atria and ventricles were investigated using standard microelectrodes, a force transducer and a video edge-detection system. It was found that: (1) ACh (at 0.001-100 microm) decreased the force of contraction and shortened the action potential duration (APD) in both atria and ventricles in a concentration-dependent manner, and that the atria were more sensitive to ACh than the ventricles; and (2) the direct negative inotropic effect of ACh (1 microm) on an isolated cardiac cell was similar to that on the isolated myocardium. But this effect was not present in all isolated ventricular cells, while all the atrial cells responded to ACh. In conclusion, ACh had direct inhibitory effects on both atrial and ventricular tissue and myocytes, although the effects were greater in atria than in ventricles; and the negative inotropic effect of ACh was closely related to the shortening of the APD.

Barium block of the muscarinic potassium current in guinea-pig atrial cells.

Block of the muscarinic K+ current (iK,ACh) by Ba2+ has been studied in guinea-pig atrial cells using the whole-cell patch-clamp technique. The dose-response curve for the block of iK,ACh can be fitted assuming that a muscarinic K+ channel is blocked when a single Ba2+ ion binds to it (apparent dissociation constant, Kd = 125 microM at 0 mV). Block was voltage and time dependent. The voltage dependence can be explained by Ba2+ binding to a site within the pore of the channel, 36% across the width of the membrane electric field (from the outside). Raising the bathing K+ concentration reduced Ba2+ block of iK,ACh, which suggests that Ba2+ and K+ compete for a common binding site. When Ba2+ was added during an exposure to ACh (muscarinic K+ channel open), block of iK,ACh developed rapidly, but when Ba2+ was added prior to an exposure to ACh (muscarinic K+ channel closed), little block of iK,ACh was evident when ACh was first applied. This suggests that when the muscarinic K+ channel is closed in the absence of ACh, Ba2+ does not have access to the binding site within the pore of the channel. In conclusion, Ba2+ block of iK,ACh is concentration, voltage, time, K+ and state dependent.

Ca2+ sparks involving multiple Ca2+ release sites along Z-lines in rat heart cells.

1. High spatial resolution confocal imaging was used to investigate the fundamental nature of "Ca2+ sparks' in rat cardiac myocytes loaded with the fluorescent calcium indicator, fluo-3. 2. The sites at which calcium sparks occurred (Ca2+ release sites) were packed closely and irregularly in transverse planes along Z-lines (mean spacing between sites of 0.76 microns). In contrast, sites were spaced more regularly in the longitudinal direction, at intervals of 1.8 microns (i.e. the sarcomere length). 3. Diffusion of released Ca2+ was slower transversely (apparent diffusion coefficient, D, 7.9 microns 2 s-1) than longitudinally (D, 17.1 microns 2 s-1). 4. Frequently, discrete sites several hundred nanometres apart transversely activated in near synchrony. The probability of transverse synchronous activity fell to low levels (< 20%) at sites separated by more than 1.0 micron. Synchronous activation was not observed between sites on different Z-lines (i.e. separated longitudinally by 1.8 microns). 5. High temporal resolution confocal microscopy (stationary spot) revealed Ca2+ sparks with "stepped' rises, consistent with multiple sites of origin. 6. We conclude that the Ca2+ spark as originally described is usually not an "elementary' event, in the sense of being indivisible, but is often comprised of yet smaller, triggered units of Ca2+ release.

ATP-dependent desensitization of the muscarinic K+ channel in rat atrial cells.

1. Fast desensitization of the muscarinic K+ channel has been studied in excised patches from rat atrial cells. 2. In inside-out patches, ACh was present in the pipette and GTP was applied via the bath to activate the channel. In outside-out patches, GTP was present in the pipette and ACh was applied via the bath to activate the channel. In both cases, during a 30 s exposure to GTP or ACh there was a decline in channel activity as a result of fast desensitization if ATP was present. 3. In inside-out patches, fast desensitization was still observed if the muscarinic ACh receptor was bypassed and the channel was activated by GTP gamma S. This suggests that fast desensitization is a result of a modification of the channel (or the connecting G protein) and not the receptor. 4. In both inside-out and outside-out patches, channel activity was depressed and fast desensitization was reduced or absent, if ATP was not present. 5. The non-hydrolysable analogue of ATP, AMP-PNP, did not substitute for ATP in its effects on the channel. 6. The results are consistent with the hypothesis that fast desensitization of the muscarinic K+ channel is the result of a dephosphorylation of the channel.

Ultra-slow voltage-dependent inactivation of the calcium current in guinea-pig and ferret ventricular myocytes.

L-type Ca2+ current, iCa, has been recorded in guinea-pig ventricular myocytes at 36 degrees C using the whole cell patch clamp technique. Intracellular Ca2+ was buffered with ethylenebis(oxonitrilo)tetraacetate (EGTA). An increase in the rate of stimulation from 0.5 to 3 Hz resulted in an abrupt decrease in iCa in the first beat at the high rate, followed by a progressive decrease (tau approx. 7 s) over the next 30 s. The changes were not the result of Ca(2+)-dependent inactivation, because similar changes occurred with either Ba2+ or Na+ as the charge carrier. During 20-s voltage clamp pulses there was an ultra-slow phase of inactivation of Ba2+ or Na+ current through the Ca2+ channel (tau approx. 6 s at 0 mV). This was confirmed by applying test pulses after conditioning pulses of different duration: the Ba2+ current during the test pulse decreased progressively when the duration of the conditioning pulse was increased progressively to 20 s. Ultra-slow inactivation of Ba2+ current was voltage dependent and increased monotonically at more positive potentials. Recovery of Ba2+ current from ultra-slow inactivation occurred with a time constant of 3.7 s at -40 mV and 0.7 s at -80 mV. The gradual decrease in iCa on increasing the rate to 3 Hz may have been the result of the development of ultra-slow voltage-dependent inactivation.

On the role of G protein activation and phosphorylation in desensitization to acetylcholine in guinea-pig atrial cells.

1. The ACh-activated K+ current (IK,ACh) has been investigated in guinea-pig atrial cells at 36 degrees C using the whole-cell patch-clamp technique. 2. During an exposure to ACh, IK,ACh faded as a result of desensitization. Throughout the fade of the current, the current reversed at EK and showed inward-going rectification. The fade was, therefore, the result of a genuine decrease in IK,ACh. 3. The onset of desensitization (as judged by the fade of IK,ACh) was biphasic and the time constants of the fast and slow phases of desensitization were 1.58 +/- 0.14 (n = 16) and 148.2 +/- 12.8 s (n = 18) respectively. Recovery from the fast and slow phases of desensitization (after 30 s and 5 min exposures to ACh respectively) occurred with time constants of 52 and 222 s respectively. This suggests that two processes are involved in desensitization. 4. The Q10 of the rate constant of the fast phase of desensitization was 2.2 +/- 0.3 (n = 6). 5. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or extracellular perfusion with AlF4- were used to bypass the muscarinic receptor and trigger IK,ACh by directly activating the G protein, GK, that links the muscarinic receptor to the K+ channel. Both GTP gamma S and AlF4- activated a current with the same reversal potential and the same degree of inward-going rectification as the ACh-activated current. 6. Desensitization still occurred when the muscarinic receptor was bypassed and IK,ACh was triggered by direct activation of GK with either GTP gamma S or AlF4-. This suggests that desensitization is, in part, the result of a modification of either GK or the K+ channel. 7. Activation of the muscarinic receptor by ACh resulted in greater desensitization than direct activation of GK; at the end of a 5 min exposure to ACh, current was only 22 +/- 1% (n = 19) of its peak value, whereas, after direct activation of GK by GTP gamma S for 5 min, current was 42 +/- 6% (n = 5) of its peak value. This suggests that desensitization also involves the muscarinic receptor. 8. When cells were perfused with GTP gamma S, the fast phase of desensitization could still occur, but the slow phase was reduced. This suggests that the fast phase involves GK or the K+ channel, whereas the slow phase involves the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization to acetylcholine in single sinoatrial node cells isolated from rabbit hearts.

The negative chronotropic effect of acetylcholine (ACh) on the sinoatrial node fades in the continuous presence of ACh as a result of desensitization. We have investigated the mechanism underlying desensitization in single rabbit sinoatrial node cells using the whole cell patch clamp technique. The negative chronotropic effect resulting from the injection of a constant hyperpolarizing current faded. ACh activated an inwardly rectifying potassium current (iK,ACh), which faded in the continuous presence of ACh. ACh had no effect on "basal" L-type calcium current (iCa), but ACh decreased iCa, which had been potentiated by isoprenaline. This effect did not fade during a 2-min exposure to ACh. ACh decreased the hyperpolarization-activated current (i(f)). This effect again did not fade. These results suggest that desensitization of the negative chronotropic response to ACh is, in part, the result of the membrane hyperpolarization and, in part, the result of the fade of iK,ACh. These results also suggest that, whereas the activation of potassium current by ACh rapidly fades, the effects resulting from the inhibition of adenylate cyclase do not.

Receptor kinase-dependent desensitization of the muscarinic K+ current in rat atrial cells.

1. Activity of rat atrial muscarinic K+ channels has been measured in five configurations of the patch clamp technique. 2. In configurations in which the normal intracellular solution was lost, the slow phase of desensitization (a slow decline of channel activity during an exposure to ACh) was much reduced (or absent) and deactivation (on wash-off of ACh) was slowed as compared with desensitization and deactivation in configurations in which normal intracellular solution was retained. This suggests that soluble intracellular regulators are involved in these processes. 3. When a G protein-coupled receptor kinase (GRK2) was applied to the cytoplasmic surface of conventional outside-out patches in the presence of ATP, the slow phase of desensitization was restored. In the absence of ATP, GRK2 failed to restore the slow phase. 4. It is concluded that (i) G protein-coupled receptor kinase dependent phosphorylation of the muscarinic receptor is responsible for the slow phase of desensitization and (ii) a soluble factor (such as a GTPase activating protein or 'GAP') is responsible for normal rapid deactivation.