High voltage-activated Ca(2+) channels of the Ca(V)1.2 class (L-type) are crucial for excitation-contraction coupling in both cardiac and smooth muscle. These channels are regulated by a variety of second messenger pathways that ultimately serve to modulate the level of contractile force in the tissue. The specific focus of this review is on the most recent advances in our understanding of how cardiac Ca(V)1.2a and smooth muscle Ca(V)1.2b channels are regulated by different kinases, including cGMP-dependent protein kinase, cAMP-dependent protein kinase, and protein kinase C. This review also discusses recent evidence regarding the regulation of these channels by protein tyrosine kinase, calmodulin-dependent kinase, purified G protein subunits, and identification of possible amino acid residues of the channel responsible for kinase regulation.

Boron is an essential mineral that plays an important role in several biological processes. Boron is required for growth of plants, animals, and humans. There are increasing evidences of this nutrient showing a variety of pleiotropic effects, ranging from anti-inflammatory and antioxidant effects to the modulation of different body systems. In the past few years, the trials showed disease-related polymorphisms of boron in different species, which has drawn attention of scientists to the significance of boron to health. Low boron profile has been related with poor immune function, increased risk of mortality, osteoporosis, and cognitive deterioration. High boron status revealed injury to cell and toxicity in different animals and humans. Some studies have shown some benefits of higher boron status, but findings have been generally mixed, which perhaps accentuates the fact that dietary intake will benefit only if supplemental amount is appropriate. The health benefits of boron are numerous in animals and humans; for instance, it affects the growth at safe intake. Central nervous system shows improvement and immune organs exhibit enhanced immunity with boron supplementation. Hepatic metabolism also shows positive changes in response to dietary boron intake. Furthermore, animals and human fed diets supplemented with boron reveal improved bone density and other benefits including embryonic development, wound healing, and cancer therapy. It has also been reported that boron affects the metabolism of several enzymes and minerals. In the background of these health benefits, low or high boron status is giving cause for concern. Additionally, researches are needed to further elucidate the mechanisms of boron effects, and determine the requirements in different species.

Abstract-Cyclic nucleotides are known to modify voltage-gated (L-type) Ca 2ϩ channel activity in vascular smooth muscle cells, but the exact mechanism(s) underlying these effects is not well defined. The purpose of the present study was to investigate the modulatory role of the cAMP-and cGMP-dependent protein kinase (PKA and PKG, respectively) pathways in Ca 2ϩ channel function by using both conventional and perforated-patch-clamp techniques in rabbit portal vein myocytes. The membrane-permeable cAMP derivative, 8-bromo cAMP (0.1 to 10 mol/L), significantly increased (14% to 16%) peak Ba 2ϩ currents, whereas higher concentrations (0.05 to 0.1 mmol/L) decreased Ba 2ϩ currents (23% to 31%). In contrast, 8-bromo cGMP inhibited Ba 2ϩ currents at all concentrations tested (0.01 to 1 mmol/L). Basal Ca V oltage-dependent (L-type) Ca 2ϩ channels play a major role in excitation-contraction coupling in vascular smooth muscle cells. L-type Ca 2ϩ channels are known to be modulated by several intracellular second-messenger systems, including both the cAMP/cAMP-dependent protein kinase (PKA) and cGMP/cGMP-dependent protein kinase (PKG) pathways.1 However, for vascular smooth muscle, little information is known regarding the exact mechanism(s) by which these processes take place. Patch-clamp studies in smooth muscle cells have shown that L-type Ca 2ϩ channel activity can be enhanced by either low concentrations of 8-Br cAMP or the catalytic subunit of PKA. 2-4Stimulation of ␤-adrenergic receptors with Iso has also been shown to increase Ca 2ϩ channel currents. 2,3,[5][6][7] On the other hand, 8-Br cGMP or the NO-releasing agents sodium nitroprusside and SNAP have been reported to lead to a decrease of Ca 2ϩ channel activity. 2,8 -10The precise mechanism underlying the effects of both PKA and PKG on L-type Ca 2ϩ channels remains controversial. A previous study from this laboratory showed that a moderate increase in cAMP elicited with 1 mol/L Iso, 1 mol/L FSK, or 0.1 mmol/L 8-Br cAMP increased Ca 2ϩ channel currents. 2On the other hand, higher levels of cAMP elicited with 10 mmol/L Iso, 10 mol/L FSK, 1 mmol/L 8-Br cAMP, or 0.1 mmol/L 8-Br cGMP led to inhibition of Ca 2ϩ channel currents. Experiments that measured the time course of responses to high concentrations of Iso or FSK revealed that Ca 2ϩ channel currents were initially enhanced and subsequently inhibited. It has been suggested that moderate increases in cAMP enhance Ca 2ϩ channel currents through PKA activation, whereas higher levels of cAMP lead to activation of PKG, which then predominates over the PKA effect (ie, cross activation of PKG by cAMP). Similar findings have been recently reported in colonic smooth muscle cells. 3 In smooth muscle cells from the basilar artery, it has been shown that exposure of inside-out patches to the catalytic subunit of PKA increased L-type Ca 2ϩ channel availability. 4 In apparent conflict with these results, Sperelakis and coworkers [11][12][13] have

The circulatory response to gram-negative sepsis and its experimental counterpart, endotoxemia, includes a profound dysfunction in myocardial contractility that is resident to the myocyte and associated with reduced systolic free intracellular Ca2+ concentration ([Ca2+]i). We explored the possibility that decreased systolic [Ca2+]iin endotoxemic myocytes is correlated with reduced L-type Ca2+ current ( I Ca,L). Ventricular myocytes were isolated from guinea pigs 4 h after an intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg). Membrane potentials and Ca2+ currents were measured using whole cell patch-clamp methods. The action potential duration of endotoxemic myocytes was significantly shorter than control values (time to 50% repolarization: LPS, 314 ± 23 ms; control, 519 ± 36 ms, P < 0.05). Correspondingly, endotoxemic myocytes demonstrated significantly reduced peak I Ca,L density (3.5 ± 0.2 pA/pF) and Ba2+current ( I Ba) density (7.3 ± 0.5 pA/pF) compared with respective values of control myocytes ( I Ca,L density 6.1 ± 0.3 pA/pF, I Ba density 11.3 ± 0.8 pA/pF; P < 0.05). Endotoxemia-induced reduction in peak I Ca,L could not be attributed to alterations in current-voltage relationships, steady-state activation and inactivation, or recovery from inactivation. The β-adrenoceptor agonist isoproterenol, but not the Ca2+ channel activator BAY K 8644, reversed the LPS-induced reduction in peak I Ca,L, cell contraction, and systolic [Ca2+]i. These data demonstrate that part of the host response to endotoxemia involves diminished sarcolemmal I Ca,L of ventricular myocytes.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes. These new antibodies produced a common prominent immunoreactive band with an apparent molecular mass of 90-92 kDa in the guinea pig heart and PASMCs, and a similar molecular mass immunoreactive band was observed in the brain from homozygous Clcn3+/+ mice but not from homozygous Clcn3-/- mice. VSOACs elicited by hypotonic cell swelling in PASMCs and guinea pig atrial myocytes were nearly completely abolished by intracellular dialysis with two new anti-ClC-3 antibodies specifically targeting the ClC-3 carboxy (C670-687 antibody) and amino terminus (A1-14 antibody). This inhibition of native VSOACs can be attributed to a specific interaction with endogenous ClC-3, because 1) preabsorption of the antibodies with corresponding antigens prevented the inhibitory effects, 2) extracellular application of a new antibody raised against an extracellular epitope (Ex133-148) of ClC-3 failed to inhibit native VSOACs in PASMCs, 3) intracellular dialysis with an antibody targeting Kv1.1 potassium channels failed to inhibit native VSOACs in guinea pig atrial myocytes, and 4) anti-ClC-3 C670-687 antibody had no effects on swelling-induced augmentation of the slow component of the delayed rectifying potassium current in guinea pig ventricular myocytes, although VSOACs in the same cells were inhibited by the antibody. These results confirm that endogenous ClC-3 is an essential molecular entity responsible for native VSOACs in PASMCs and guinea pig cardiac myocytes.

Electroacupuncture (EA) has been used for treating visceral hypersensitivity (VH). However, the underlying molecular mechanism remains unclear. This study was aim to testify the effect of EA on ileitis-provoked VH, and to confirm whether EA attenuates VH through Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathway in the periaqueductal gray (PAG)-the rostral ventromedial medulla (RVM)-the spinal cord dorsal horn (SCDH) axis. Methods:Goats were anesthetized and laparotomized for injecting 2,4,6-trinitro-benzene-sulfonic acid (TNBS)-ethanol solution (30 mg TNBS dissolved in 40% ethanol) into the ileal wall to induce VH. EA was treated for 30 min from day 7, then every 3 days for six times. VH was assessed by visceromotor response (VMR) and pain behavior response to 20, 40, 60, 80, and 100 mmHg colorectal distension pressures at day 7, 10, 13, 16, 19, and 22. The spinal cord in the eleventh thoracic vertebra and the brain were collected at day 22. The protein and mRNA levels of IL-6, JAK2, and STAT3 in the SCDH were detected with western blot and qPCR, respectively. The distribution of these substances was observed with immunohistochemistry in the ventrolateral PAG (vlPAG), RVM (mainly the nucleus raphe magnus, NRM), SCDH, the nucleus tractus solitaries (NTS) and the dorsal motor nucleus of vagi (DMV).Results: Goats administered with TNBS-ethanol solution showed diarrhea, enhanced VMR and pain behavior response, and increased IL-6, phosphorylated JAK2 and STAT3 (pJAK2 and pSTAT3) in the vlPAG, NRM, NTS and DMV, and their protein and mRNA levels in the SCDH. EA relieved diarrhea, VMR and pain behavior response, decreased IL-6, pJAK2 and pSTAT3 levels in the vlPAG, NRM, SCDH, NTS, and DMV except for pSTAT3 in the DMV, but did not affect mRNA level of these three substances in the SCDH.Conclusion: EA attenuates VH probably through inhibiting JAK2/STAT3 signaling pathway in the PAG-RVM-SCDH axis.

Previous studies have demonstrated the protective signaling of hypoxia-inducible factor (HIF)-1 ␣ against ischemia-reperfusion (I/R) injury in the heart. In the present study, we provide further evidence for a cardioprotective mechanism by HIF-1␣ against I/R injury exerted via the mitochondrial protein frataxin, which regulates mitochondrial Fe-S cluster formation. Disruption of frataxin has been found to induce mitochondrial iron overload and subsequent ROS production. We observed that frataxin expression was elevated in mice hearts subjected to I/R injury, and this response was blunted in cardiomyocyte-specific HIF-1␣ knockout (KO) mice. Furthermore, these HIF-1␣ KO mice sustained extensive cardiac damage from I/R injury compared with control mice. Similarly, reduction of HIF-1␣ by RNA inhibition resulted in an attenuation of frataxin expression in response to hypoxia in H9C2 cardiomyocytes. Therefore, we postulated that HIF-1␣ transcriptionally regulates frataxin expression in response to hypoxia and offers a cardioprotective mechanism against ischemic injury. Our promoter activity and chromatin immunoprecipitation assays confirmed the presence of a functional hypoxia response element in the frataxin promoter. Our data also suggest that increased frataxin mitigated mitochondrial iron overload and subsequent ROS production, thus preserving mitochondrial membrane integrity and viability of cardiomyocytes. We postulate that frataxin may exert its beneficial effects by acting as an iron storage protein under hypoxia and subsequently facilitates the maintenance of mitochondrial membrane potential and promotes cell survival. The findings from our study revealed that HIF-1␣-frataxin signaling promotes a protective mechanism against hypoxic/ischemic stress.hypoxia-inducible factor-1␣; frataxin; iron-sulfur; mitochondria; ischemia-reperfusion NEWS & NOTEWORTHYThe present study provides evidence for a cardioprotective transcriptional regulatory mechanism by hypoxia-inducible factor-1␣ of the mitochondrial protein frataxin against ischemia-reperfusion injury. Frataxin regulates mitochondrial Fe-S cluster formation and protects against mitochondrial iron overload, the subsequent ROS production, and myocardial energy dysregulation.