Thermosensitivity of the voltage-dependent activation of calcium homeostasis modulator 1 (calhm1) ion channel

Calcium homeostasis modulator 1 (calhm1) proteins form an outwardly rectifying nonselective ion channel having exceedingly slow kinetics and low sensitivity to voltage that is shifted by lowering extracellular Ca2+ ([Ca2+]e). Here we found that physiological temperature dramatically facilitates the voltage-dependent activation of the calhm1 current (Icalhm1); increased amplitude (Q10, 7-15) and fastened speed of activation. Also, the leftward shift of the half-activation voltage (V1/2) was similary observed in the normal and lower [Ca2+]e. Since calhm1 is highly expressed in the brain and taste cells, the thermosensitivity should be considered in their electrophysiology.

Alteration of the mu opioid receptor: Ca2+ channel signaling pathway in a subset of rat sensory neurons following chronic femoral artery occlusion

The exercise pressor reflex, a crucial component of the cardiovascular response under physiological and pathophysiological states, is activated via metabolic and mechanical mediators that originate from contracting muscles and stimulate group III and IV afferents. We reported previously that stimulation of mu opioid receptors (MOR), expressed in both afferents, led to a significant attenuation of the reflex in rats whose femoral arteries had been occluded for 72 h. The present study examined the effect of arterial occlusion on the signaling components involved in the opioid-mediated modulation of Ca2+ channels in rat dorsal root ganglion neurons innervating the triceps surae muscles. We focused on neurons that were transfected with cDNA coding for enhanced green fluorescent protein whose expression is driven by the voltage-gated Na+ channel 1.8 (NaV1.8) promoter region, a channel expressed primarily in nociceptive neurons. With the use of a small interference RNA approach, our results show that the pertussis toxin-sensitive Gαi3 subunit couples MOR with Ca2+ channels. We observed a significant leftward shift of the MOR agonist [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin concentration-response relationship in neurons isolated from rats with occluded arteries compared with those that were perfused freely. Femoral occlusion did not affect Ca2+ channel density or the fraction of the main Ca2+ channel subtype. Furthermore, Western blotting analysis indicated that the leftward shift did not result from either increased Gαi3 or MOR expression. Finally, all neurons from both groups exhibited an inward current following exposure of the transient potential receptor vanilloid 1 (TRPV1) agonist, 8-methyl-N-vanillyl-6-nonenamide. These findings suggest that sensory neurons mediating the exercise pressor reflex express NaV1.8 and TRPV1 channels, and femoral occlusion alters the MOR pharmacological profile.

Neonatal maternal deprivation sensitizes voltage-gated sodium channel currents in colon-specific dorsal root ganglion neurons in rats

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain in association with altered bowel movements. The underlying mechanisms of visceral hypersensitivity remain elusive. This study was designed to examine the role for sodium channels in a rat model of chronic visceral hyperalgesia induced by neonatal maternal deprivation (NMD). Abdominal withdrawal reflex (AWR) scores were performed on adult male rats. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch-clamp configurations. The expression of NaV1.8 was analyzed by Western blot and quantitative real-time PCR. NMD significantly increased AWR scores, which lasted for ∼6 wk in an association with hyperexcitability of colon DRG neurons. TTX-resistant but not TTX-sensitive sodium current density was greatly enhanced in colon DRG neurons in NMD rats. Compared with controls, activation curves showed a leftward shift in NMD rats whereas inactivation curves did not differ significantly. NMD markedly accelerated the activation time of peak current amplitude without any changes in inactivation time. Furthermore, NMD remarkably enhanced expression of NaV1.8 at protein levels but not at mRNA levels in colon-related DRGs. The expression of NaV1.9 was not altered after NMD. These data suggest that NMD enhances TTX-resistant sodium activity of colon DRG neurons, which is most likely mediated by a leftward shift of activation curve and by enhanced expression of NaV1.8 at protein levels, thus identifying a specific molecular mechanism underlying chronic visceral pain and sensitization in patients with IBS.

Potentiation of NO-dependent activation of soluble guanylyl cyclase by 5-nitroisatin and antiviral preparatation arbidol

Isatin (indole-dione) is an endogenous indole that exibits a wide range of biological and physiological activity. The influence of isatin derivatives 5-nitroisatin and arbidol (an antiviral preparatation) on spermine NONO-induced activation of human platelet soluble guanylyl cyclase was investigated. 5-nitroistnin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylyl cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. At the same time both compounds used did not influence the activation of guanylyl cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylyl cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylyl cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.

Pharmacological Basis for the Medicinal Use ofLepidium sativumin Airways Disorders

Lepidium sativumis widely used in folk medicine for treatment of hyperactive airways disorders, such as asthma, bronchitis and cough. The crude extract ofLepidium sativum(Ls.Cr) inhibited carbachol (CCh, 1 μM-) and K+(80 mM-) induced contractions in a pattern similar to that of dicyclomine. Ls.Cr at 0.03 mg/mL produced a rightward parallel shift of CCh curves, followed by nonparallel shift at higher concentration (0.1 mg/mL), suppressing maximum response, similar to that caused by dicyclomine. Pretreatment of tissues with Ls.Cr (0.1–0.3 mg/mL) shifted Ca++concentration-response curves (CRCs) to right, as produced by verapamil. Ls.Cr at low concentrations (0.03–0.1 mg/mL) caused leftward shift of isoprenaline-induced inhibitory CRCs, like that caused by rolipram, a phosphodiesterase (PDE) inhibitor. These results indicate that bronchodilatory effect ofLepidium sativumis mediated through a combination of anticholinergic, Ca++antagonist and PDE inhibitory pathways, which provides sound mechanistic background for its medicinal use in the overactive airways disorders.

Arterial [H+] and the ventilatory response to hypoxia in humans: influence of acetazolamide-induced metabolic acidosis

In this study, we investigated possible separate effects of H+ ions and CO2 on hypoxic sensitivity in humans. We also examined whether hypoxic sensitivity, conventionally defined as the ratio of (hypoxic − normoxic) ventilation over (hypoxic − normoxic) Hb oxygen saturation can also be estimated by taking the ratio (hypoxic − normoxic) ventilation over (logPaO2 hypoxia − logPaO2 normoxia), enabling one to measure the hypoxic response independently from potential confounding influences of changes in position of the Hb oxygen saturation curve. We used acetazolamide to induce a metabolic acidosis. To determine the acute hypoxic response (AHR), we performed step decreases in end-tidal Po2 to ∼50 Torr lasting 5 min each at three different constant end-tidal Pco2 levels. Nine subjects ingested 250 mg of acetazolamide or placebo every 8 h for 3 days in a randomized double-blind crossover design. The metabolic acidosis was accompanied by a rise in ventilation, a substantial fall in PaCO2, and a parallel leftward shift of the ventilatory CO2 response curve. In placebo, CO2 induced equal relative increases in hypoxic sensitivity (O2-CO2 interaction) regardless of the way it was defined. Acetazolamide shifted the response line representing the relationship between hypoxic sensitivity and arterial [H+] ([H+]a) to higher values of [H+]a without altering its slope, indicating that it did not affect the O2-CO2 interaction. So, in contrast to an earlier belief, CO2 and H+ have separate effects on hypoxic sensitivity. This was also supported by the finding that infusion of bicarbonate caused a leftward shift of the hypoxic sensitivity-[H+]a response lines in placebo and acetazolamide. A specific inhibitory effect of acetazolamide on hypoxic sensitivity was not demonstrated.

β-Adrenergic relaxation of mouse urinary bladder smooth muscle in the absence of large-conductance Ca2+-activated K+ channel

In urinary bladder smooth muscle (UBSM), stimulation of β-adrenergic receptors (β-ARs) leads to activation of the large-conductance Ca2+-activated K+ (BK) channel currents (Petkov GV and Nelson MT. Am J Physiol Cell Physiol 288: C1255–C1263, 2005). In this study we tested the hypothesis that the BK channel mediates UBSM relaxation in response to β-AR stimulation using the highly specific BK channel inhibitor iberiotoxin (IBTX) and a BK channel knockout (BK-KO) mouse model in which the gene for the pore-forming subunit was deleted. UBSM strips isolated from wild-type (WT) and BK-KO mice were stimulated with 20 mM K+ or 1 μM carbachol to induce phasic and tonic contractions. BK-KO and WT UBSM strips pretreated with IBTX had increased overall contractility, and UBSM BK-KO cells were depolarized with ∼12 mV. Isoproterenol, a nonspecific β-AR agonist, and forskolin, an adenylate cyclase activator, decreased phasic and tonic contractions of WT UBSM strips in a concentration-dependent manner. In the presence of IBTX, the concentration-response curves to isoproterenol and forskolin were shifted to the right in WT UBSM strips. Isoproterenol- and forskolin-mediated relaxations were enhanced in BK-KO UBSM strips, and a leftward shift in the concentration-response curves was observed. The leftward shift was eliminated upon PKA inhibition with H-89, suggesting upregulation of the β-AR-cAMP pathway in BK-KO mice. These results indicate that the BK channel is a key modulator in β-AR-mediated relaxation of UBSM and further suggest that alterations in BK channel expression or function could contribute to some pathophysiological conditions such as overactive bladder and urinary incontinence.

The Role of the Cerebellum in Subsecond Time Perception: Evidence from Repetitive Transcranial Magnetic Stimulation

In three experiments, we investigated the role of the cerebellum in sub- and suprasecond time perception by using repetitive transcranial magnetic stimulation (rTMS). In Experiment 1, subjects underwent four 8-min 1-Hz rTMS sessions in a within-subject design. rTMS sites were the medial cerebellum (real and sham rTMS), left lateral cerebellum, and right lateral cerebellum. Following each rTMS session, subjects completed a subsecond temporal bisection task (stimuli in the range 400–800 msec). Compared with sham rTMS, rTMS applied over the right lateral or medial cerebellum induced a leftward shift of the psychophysical function (perceived lengthening of time). In Experiment 2, a separate sample of subjects underwent the identical rTMS procedure and completed a suprasecond bisection task (stimuli in the 1000–2000 msec range). In this experiment, rTMS to the cerebellar sites did not produce any significant changes compared with sham rTMS. Experiment 3 employed a within-subject design to replicate findings from Experiments 1 and 2. Subjects underwent four rTMS conditions (sub- and suprabisection tasks following medial cerebellar and sham rTMS). rTMS induced a significant leftward shift of psychophysical function in the subsecond bisection, but not in the suprasecond bisection. In this study, we have demonstrated that transient cerebellar stimulation can differently affect the ability to estimate time intervals below and above a duration of 1 sec. The results of this study provide direct evidence for the role of the cerebellum in processing subsecond time intervals. This study further suggests that the perception of sub- and suprasecond intervals is likely to depend upon distinct neural systems.