KCl-induced Increase Research Articles

Pu-Erh Tea Relaxes the Thoracic Aorta of Rats by Reducing Intracellular Calcium.

Previous studies suggested that pu-erh tea aqueous extract could lower blood pressure and ameliorate hypertension symptoms. However, the antihypertension mechanisms of pu-erh tea remain unclear. In this work, the direct effects of pu-erh tea on vessels and cells were investigated by detecting isometric tension and intracellular calcium ([Ca2+]i), respectively. Additionally, to identify the main active components, the aqueous extract of pu-erh was separated by organic solvents to obtain various fractions, and the effects of these fractions on arteries were assessed. The results showed that pu-erh aqueous extract vasodilated rat thoracic aortas preconstricted by phenylephrine or KCl. These vasodilation effects were not significantly affected by the removal of the endothelium or by preincubation with potassium channel blockers (tetraethylammonium, glibenclamide, aminopyridine, or barium chloride). Moreover, pu-erh aqueous extract could reduce the vessel contractibility induced by CaCl2 and phenylephrine under KCl-depolarizing or Ca2+-free buffer conditions, respectively. Furthermore, pu-erh aqueous extract attenuated the KCl-induced increase in [Ca2+]i in cultured rat aortic smooth muscle A7r5 cells. In addition, the chloroform precipitate of pu-erh aqueous extract produced the most potent vasodilation. Theabrownins (the characteristic components of pu-erh tea) accounted for 41.91 ± 1.09 % of the chloroform precipitate and vasodilated arteries in an endothelium-independent manner. In addition, the vasodilation effect of caffeine was verified. In conclusion, theabrownins and caffeine should be the two main active components in pu-erh tea. Pu-erh aqueous extract vasodilated arteries in an endothelium-independent manner, which might partly be attributed to the decrease in extracellular Ca2+ influx. Moreover, our study provided data on the potential mechanism of the hypotensive actions of pu-erh tea, which might improve our understanding of the effect of pu-erh tea on the prevention and treatment of hypertension.

A New Model of Sensorial Neuron-Like Cells for HTS of Novel Analgesics for Neuropathic Pain

In this study we developed a new translational phenotypic in vitro model for high-throughput screening (HTS) of novel analgesics for treating neuropathic pain, in order to address the poor translation of traditional recombinant models. The immortalized dorsal root ganglia (DRG) neuron-like F11 cell line was selected based on its phenotype after differentiation. The acquisition of neuronal characteristics was evaluated by measuring the expression of TrkA as a DRG neuron marker (p < 0.01) as well as by measuring the global neurite length (p < 0.001). The response of F11 cells to ATP and KCl was obtained by measuring intracellular calcium concentration, dynamic mass redistribution, and membrane potential. A KCl-induced increase of intracellular calcium levels was chosen as the readout because of the better signal quality, higher reproducibility, and greater compatibility with HTS assay requirements compared with other methods. The response to KCl differed significantly between differentiated and undifferentiated cells (p < 0.05), with an EC50 value of 5 mM in differentiated cells. The model was validated by screening the Prestwick Chemical Library. Five hits already proposed for neuropathic-related pain were identified, with IC50 values between 1 and 7 µM. This cell model provides a new tool for screening novel analgesics for the relief of neuropathic pain.

Rho Kinase Inhibitors Are Involved in L‐Type Voltage‐dependent Calcium Channel Pathway in Vascular Smooth Muscle Cells

Voltage‐dependent L‐type calcium channels (L‐VDCC) and the rho/rho kinase pathway are two predominate intracellular pathways that regulate renal microvascular tone and reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests these pathways are convergent. We hypothesized that the rho kinase inhibitors influenced L‐VDCC signaling. The effects of rho kinase inhibitors Y‐27632 or RKI‐1447 on KCl‐depolarization were assessed in afferent arterioles using the in vitro blood‐perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30, 60, and 90mM) led to concentration‐dependent vasoconstriction of afferent arterioles reducing the diameter to 95±6, 56±4 and 42±3% of the baseline, respectively (p&lt;0.05). Addition of Y‐27632 (1, 5 and 10 μM) or RKI‐1447 (0.1, 1 and 10 μM) significantly increased starting diameter by 16–65% (n=6/each, p&lt;0.05). The KCl‐induced vasoconstriction with 1 μM Y‐27632 or 0.1 and 1 μM RKI‐1447 was similar to control, but markedly attenuated with 5 and 10 μM Y‐27632, and 10 μM RKI‐1447 (p&lt;0.05 vs KCl alone). The afferent arteriole diameter averaged 99±2, 83±2, 71±4%, and 98±0.8, 89±5, 81±6% of the baseline for 5 and 10 μM Y‐27632, and 97±2, 89±3 and 88±4% of the baseline for 10 μM RKI‐1447, respectively (n=6/each, p&lt;0.05 vs KCl alone). Changes in intracellular calcium concentration [Ca2+]i were estimated by fura‐2‐AM fluorescence (ratio F340/380) during KCl‐depolarization in cultured A7r5 cells (n=46–118/each group) and in freshly isolated renal microvascular smooth muscle cells (RMVSMCs, n=32–118/each group). Administration of 90 mM KCl increased fura‐2 fluorescence markedly in both cell types. The KCl‐induced increase of the F340/F380 ratio was unaltered by 0.1 or 0.5 μM Y‐27632 but was significantly attenuated by Y‐27632 between 1–10 μM by 50–56% (p&lt;0.05 vs KCl alone) in A7r5 cells but only at 10 μM in RMVSMCs by 42% (p&lt;0.05 vs KCl alone). RKI‐1447 however, significantly attenuated KCl‐mediated elevation of [Ca2+]i in both cell types regardless of the concentrations applied (0.1–10 μM, p&lt;0.05), indicating that Y‐27632 and RKI‐1447 decrease L‐VDCC function. These studies indicate that the rho kinase inhibitors, Y‐27632 and RKI‐1447 partially inhibit L‐VDCC function and participate in L‐VDCC signaling.Support or Funding InformationThis study was supported by a Grant‐in‐Aid (15GRNT25240015) from the American Heart Association Great Southeast Affiliate and a grant from NIH (DK106500) to ZG, and by grants from NIH (DK044628, HL074167, HL098135 and HL095499) to EWI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of orexin receptor subtypes in the inhibitory effects of orexin-A on potassium chloride-induced increases in intracellular calcium ion levels in neurons derived from dorsal root ganglion of carrageenan-treated rats

We analysed the roles of orexin receptors in the effects of orexin-A on KCl-induced increases in intracellular calcium ion levels ([Ca2+]i) in C-fiber-like small neurons of rats with inflammation induced by intraplantar injection of carrageenan into the hind paw. Controls were treated with saline. Paw withdrawal and threshold forces in response to tactile stimuli were determined using von Frey filaments. [Ca2+]i in C-fiber-like neurons derived from dorsal root ganglia was visualised using a calcium fluorescence probe. Changes in neuronal [Ca2+]i were assessed as relative fluorescence intensity (F/F0). One day after carrageenan injection, the paw withdrawal response to tactile stimuli and the paw withdrawal threshold were increased and reduced, respectively. KCl loading of neurons from either carrageenan-treated or control rats increased F/F0 to about 2.0. KCl-induced increases in F/F0 of carrageenan-treated, but not control, rats were inhibited by orexin-A. The OX1 and OX2 receptor antagonist MK-4305, but not the OX1 receptor antagonist SB334867, counteracted the effects of orexin-A on the KCl-induced increase in F/F0. These results suggest that OX2, but not OX1 receptors mediate the inhibitory effect of orexin-A on KCl-induced increases in [Ca2+]i in C-fiber-like neurons of rats with inflammation.

Paeoniflorin, the Main Active Ingredient of Shuyu Capsule, Inhibits Cav1.2 and Regulates Calmodulin/Calmodulin-Dependent Protein Kinase II Signalling.

The aim of this study was to explore the mechanism underlying the antidepression activity of paeoniflorin, the main active ingredient of paeony extract and Shuyu capsules, and determine its effect on the calmodulin/calmodulin-dependent protein kinase II (CaM/CaMKII) signalling pathway and on the possible target, the voltage-gated calcium channel (Cav). Rats at the nonacceptance stage were selected for premenstrual syndrome (PMS) depression modelling. Behavioural assays were used for model testing. Rats were given Shuyu capsules, paeony extract, and bupleurum. Western blot analysis was used to assess the expression levels of calcium voltage-gated channel subunit alpha 1 C (CACNA1C), brain-derived neurotrophic factor, and CaM/CaMKII signalling pathway proteins. Intracellular Ca2+ concentration in CHO cell line was measured using Fluo-4-AM and whole-cell patch clamps. The PMS depression model was successfully established and demonstrated that Shuyu can mitigate depressive behaviour in a rat PMS model. Paeony extract did not affect CACNA1C protein expression in rat hippocampi but did affect Cav1.2-mediated CaM/CaMKII signalling pathways. Paeoniflorin significantly inhibited KCl-induced increases in intracellular Ca2+ concentration and Cav1.2 current density. Further, it may function via the CaM/CaMKII pathway and its downstream signalling molecules by regulating Cav1.2, thus playing an important role in the treatment and alleviation of affective disorders.

Menthol inhibits detrusor contractility independently of TRPM8 activation.

Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25–30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM–30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.

Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150μg/50μL intraplantarly) and D-Ala2-MePhe4-Glyol5 enkephalin (300μg/50μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5mg/kg intraperitoneally) and MK-801 (0.2mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3mg/kg subcutaneously), but not loperamide (3mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca2+]i. However, this drug effect significantly decreased in cells pretreated with loperamide (3μM, 72hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide’s inhibition of KCl-elicited [Ca2+]i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.

Modification of Ca2+-handling in cardiomyocytes by redox sensitive mechanisms in response to ouabain

We examined the role of redox-sensitive signal transduction mechanisms in modifying the changes in [Ca(2+)](i) produced by ouabain upon incubating adult rat cardiomyocytes with antioxidants or inhibitors of different protein kinases and monitoring alterations in fura-2 fluorescence. Ouabain increased basal [Ca(2+)](i), augmented the KCl-induced increase in [Ca(2+)](i), and promoted oxyradical production in cardiomyocytes. These actions of ouabain were attenuated by an oxyradical scavenging mixture (superoxide dismutase plus catalase), and the antioxidants (N-acetyl-L-cysteine and N-(2-mercaptoproprionyl)glycine). An inhibitor of MAP kinase (PD98059) depressed the ouabain-induced increase in [Ca(2+)], whereas inhibitors of tyrosine kinase (tyrphostin and genistein) and PI3 kinase (Wortmannin and LV294002) enhanced the ouabain-induced increase in [Ca(2+)](i). Inhibitors of protein kinase C (calphostin and bisindolylmalaimide) augmented the ouabain-induced increase in [Ca(2+)](i), whereas stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate) depressed the action of ouabain. These results suggest that ouabain-induced inhibition of Na (+)-K(+) ATPase may alter the redox status of cardiomyocytes through the production of oxyradicals, and increase the activities of various protein kinases. Thus, these redox-sensitive signal transduction mechanisms involving different protein kinases may modify Ca(2+)-handling sites in cardiomyocytes and determine the magnitude of net increase in [Ca(2+)](i) in response to ouabain.

Mechanisms of the beneficial effects of vitamin B6 and pyridoxal 5-phosphate on cardiac performance in ischemic heart disease

Although vitamin B6 and its metabolite, pyridoxal 5'-phosphate (PLP), have been shown to exert beneficial effects in ischemic heart disease, the mechanisms of their action are not fully understood. Some studies have shown that ventricular arrhythmias and mortality upon the occlusion of coronary artery were attenuated by pretreatment of animals with PLP. Furthermore, ischemia-reperfusion-induced abnormalities in cardiac performance and defects in sarcoplasmic reticular Ca2+-transport activities were decreased by PLP. The increase in cardiac contractile activity of isolated heart by ATP was reduced by PLP, unlike propranolol, whereas that by isoproterenol was not depressed by PLP. ATP-induced increase in [Ca2+]i, unlike KCl-induced increase in [Ca2+]i in cardiomyocytes was depressed by PLP. Both high- and low-affinity sites for ATP binding in sarcolemmal membranes were also decreased by PLP. These observations support the view that PLP may produce cardioprotective effects in ischemic heart disease by attenuating the occurrence of intracellular Ca2+ overload due to the blockade of purinergic receptors.

Sigma-1 receptors do not regulate calcium influx through voltage-dependent calcium channels in mouse brain synaptosomes

Several lines of evidence suggest that σ1 receptors regulate intracellular calcium concentration [Ca2+]i. However, no previous studies have demonstrated a consistent role for these receptors in the modulation of extracellular calcium entry through plasmalemmal voltage-dependent calcium channels (VDCCs). To search for evidence of such a role we compared [Ca2+]i under basal conditions and after depolarization with KCl in fura-2-loaded synaptosomes from wild-type and σ1 receptor knockout (σ1R-KO) mice. We also tested the effects of the selective σ1 receptor agonists PRE-084 and (+)-pentazocine and antagonists BD-1047 and NE-100 on the increase in [Ca2+]i induced by depolarization with 60mM KCl. Mibefradil, a nonselective blocker of VDCCs, was used as a positive control. Basal [Ca2+]i and the increase in [Ca2+]i caused by KCl-induced depolarization were similar in brain synaptosomes from both wild-type and σ1R-KO mice. Mibefradil (1–30μM) and all σ1 receptor ligands studied (3–100μM) inhibited the KCl-induced increase in [Ca2+]i in a concentration-dependent way. The order of maximum inhibition for the ligands compared here was NE-100>BD-1047=PRE 084>(+)-pentazocine. There were no appreciable differences in their effects between wild-type and σ1R-KO mice. These findings indicate that σ1 receptors are not involved in calcium influx through VDCCs or in the inhibitory effects of these σ1 ligands on Ca2+ channels.

Mechanism of glucose-6-phosphate dehydrogenase-mediated regulation of coronary artery contractility

We previously identified glucose-6-phosphate dehydrogenase (G6PD) as a regulator of vascular smooth muscle contraction. In this study, we tested our hypothesis that G6PD activated by KCl via a phosphatase and tensin homologue deleted on chromosome 10 (PTEN)-protein kinase C (PKC) pathway increases vascular smooth muscle contraction and that inhibition of G6PD relaxes smooth muscle by decreasing intracellular Ca(2+) ([Ca(2+)](i)) and Ca(2+) sensitivity to the myofilament. Here we show that G6PD is activated by membrane depolarization via PKC and PTEN pathway and that G6PD inhibition decreases intracellular free calcium ([Ca(2+)](i)) in vascular smooth muscle cells and thus arterial contractility. In bovine coronary artery (CA), KCl (30 mmol/l) increased PKC activity and doubled G6PD V(max) without affecting K(m). KCl-induced PKC and G6PD activation was inhibited by bisperoxo(pyridine-2-carboxyl)oxovanadate (Bpv; 10 μmol/l), a PTEN inhibitor, which also inhibited (P < 0.05) KCl-induced CA contraction. The G6PD blockers 6-aminonicotinamide (6AN; 1 mmol/l) and epiandrosterone (EPI; 100 μmol/l) inhibited KCl-induced increases in G6PD activity, [Ca(2+)](i), Ca(2+)-dependent myosin light chain (MLC) phosphorylation, and contraction. Relaxation of precontracted CA by 6AN and EPI was not blocked by calnoxin (10 μmol/l), a plasma membrane Ca(2+) ATPase inhibitor or by lowering extracellular Na(+), which inhibits the Na(+)/Ca(2+) exchanger (NCX), but cyclopiazonic acid (200 μmol/l), a sarcoplasmic reticulum Ca(2+) ATPase inhibitor, reduced (P < 0.05) 6AN- and EPI-induced relaxation. 6AN also attenuated phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Ser855, a site phosphorylated by Rho kinase, inhibition of which reduced (P < 0.05) KCl-induced CA contraction and 6AN-induced relaxation. By contrast, 6AN increased (P < 0.05) vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239, indicating that inhibition of G6PD increases PKA or PKG activity. Inhibition of PKG by RT-8-Br-PET-cGMPs (100 nmol/l) diminished 6AN-evoked VASP phosphorylation (P < 0.05), but RT-8-Br-PET-cGMPs increased 6AN-induced relaxation. These findings suggest G6PD inhibition relaxes CA by decreasing Ca(2+) influx, increasing Ca(2+) sequestration, and inhibiting Rho kinase but not by increasing Ca(2+) extrusion or activating PKG.

Involvement of sarcoplasmic reticulum in changing intracellular calcium due to Na+/K+-ATPase inhibition in cardiomyocytes

Earlier studies have demonstrated that ouabain-induced increase in [Ca2+]i, as a consequence of sarcolemma (SL) Na+/K+-ATPase inhibition, is associated with activation of both the SL Na+/Ca2+ exchanger and SL Ca2+ channels. In view of the importance of sarcoplasmic reticulum (SR) in the regulation of [Ca2+]i, this study examined the role of SR in ouabain-induced increase in [Ca2+]i in both quiescent and KCl-depolarized cardiomyocytes. For this purpose, adult rat cardiomyocytes were loaded with fura-2 and ouabain-induced changes in [Ca2+]i were monitored upon treatment with or without different agents that are known to influence Ca2+ handling by the intracellular organelles. Ouabain not only increased the basal [Ca2+]i and augmented KCl-induced increase in [Ca2+]i but also produced similar effects on the ATP-induced increase in [Ca2+]i. Treatments of cardiomyocytes with caffeine, ryanodine, or cyclopiazonic acid, which affect SR Ca2+ stores, attenuated the ouabain-induced increase in basal Ca2+ as well as augmentation of the KCl response. Both ryanodine and cyclopiazonic acid produced additional effects, when used in combination with a SL Ca2+ channel inhibitor (verapamil), but not with a Na+/Ca2+ exchange inhibitor (KB-R7943). Inhibitors of Ca2+/calmodulin kinase, protein kinase A, and inositol-3-phosphate receptors were also observed to depress the ouabain-induced increase in [Ca2+]i in cardiomyocytes. On the other hand, mitochondrial Ca2+ transport inhibitors did not exert any effect on the ouabain-induced alterations in [Ca2+]i in cardiomyocytes. Furthermore, ouabain did not show any direct effect on the Ca2+ uptake and Ca2+ release activities of SR or mitochondria. These results suggest an indirect involvement of SR Ca2+ stores in the ouabain-induced increase in [Ca2+]i in cardiomyocytes and indicate the participation of both Ca2+-induced Ca2+ release and regulatory mechanisms in this action.

Cellular calcium regulatory machinery of vasorelaxation elicited by petasin

1. The present study examined the cytosolic Ca(2+) regulatory machinery involved in the vasorelaxation produced by petasin, a sesquiterpene isolated from Petasites formosanus. 2. Aortic rings isolated from Sprague-Dawley rats were exposed to petasin (0.01-100 micromol/L) to elucidate its vascular effects on isometric contraction elicited by vasoconstrictors, as well as the contribution of the endothelium and Ca(2+) to the responses observed. In addition, L-type voltage-dependent Ca(2+) channel (VDCC) activity and [Ca(2+)](i) were determined in cultured vascular smooth muscle cells (VSMCs) from Sprague-Dawley rats in the presence of 1-100 micromol/L petasin using whole-cell patch-clamp recording and the fluorescent probe fura-2/AM. The effects of petasin on vascular responses were compared between aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. 3. Petasin reduced isometric contraction elicited by KCl or the L-type Ca(2+) channel opener BayK 8644 (IC(50) 3.0 +/- 0.4 and 4.1 +/- 1.1 micromol/L, respectively) in aortic rings isolated from Sprague-Dawley rats, independent of the endothelium. In addition, petasin triggered a rightward shift in the concentration-response curve to KCl while reducing the maximal response by 82%. In Ca(2+)-depleted and high K(+)-depolarized aortic rings, 1-100 micromol/L petasin pretreatment attenuated the Ca(2+)-induced contraction in a concentration-dependent manner. 4. In cultured VSMCs, whole-cell patch-clamp recording revealed that petasin inhibited VDCC activity. Measurement of [Ca(2+)](i) using fura-2/AM fluorescence indicated that petasin suppressed the KCl-induced increase in [Ca(2+)](i). However, receptor binding assays failed to identify any significant interaction between petasin and the dihydropyridine binding sites of the L-type VDCC. 5. In aortic rings from SHR and WKY rats, petasin inhibited Ca(2+)-induced contractions in Ca(2+)-depleted and high K(+)-depolarized solution with a more pronounced effect in rings from SHR. 6. Together, the results suggest that direct Ca(2+) antagonism of L-type VDCC in vascular smooth muscle may account, at least in part, for petasin-induced vasorelaxation. The more pronounced effect of the sesquiterpene in blood vessels from SHR suggests its possible therapeutic potential in the mangement of hypertension.

Pimaradienoic acid inhibits vascular contraction and induces hypotension in normotensive rats

The present investigation was designed to investigate the effect of the diterpene ent-pimara-8(14),15-dien-19-oic acid (pimaradienoic acid, PA) on smooth muscle extracellular Ca(2+) influx. To this end, the effect of PA on phenylephrine- and KCl-induced increases in cytosolic calcium concentration ([Ca(2+)](c)), measured by the variation in the ratio of fluorescence intensities (R340/380 nm) of Fura-2, was analysed. Whether bolus injection of PA could induce hypotensive responses in conscious normotensive rats was also evaluated. PA inhibited the contraction induced by phenylephrine (0.03 or 10 micromol L(-1)) and KCl (30 or 90 mmol L(-1)) in endothelium-denuded rat aortic rings in a concentration dependent manner. Pre-treatment with PA (10, 100, 200 micromol L(-1)) attenuated the contraction induced by CaCl(2) (0.5 nmol L(-1) or 2.5 mmol L(-1)) in denuded rat aorta exposed to Ca(2+)-free medium containing phenylephrine (0.1 micro mol L(-1)) or KCl (30 mmol L(-1)). Interestingly, the inhibitory effect displayed by PA on CaCl(2)-induced contraction was more pronounced when KCl was used as the stimulant. Phenylephrine- and KCl-induced increases in [Ca(2+)](c) were inhibited by PA. Similarly, verapamil, a Ca(2+)-channel blocker, also inhibited the increase in [Ca(2+)](c) induced by either phenylephrine or KCl. Finally, bolus injection of PA (1-15 mg kg(-1)) produced a dose-dependent decrease in mean arterial pressure in conscious normotensive rats. The results provide the first direct evidence that PA reduces vascular contractility by reducing extracellular Ca(2+) influx through smooth muscle cellular membrane, a mechanism that could mediate the hypotensive response induced by this diterpene in normotensive rats.

Attenuation of ischemia–reperfusion-induced alterations in intracellular Ca2+in cardiomyocytes from hearts treated withN-acetylcysteine andN-mercaptopropionylglycineThis article is one of a selection of papers published in a special issue on Advances in Cardiovascular Research.

This study was undertaken to test whether Ca(2+)-handling abnormalities in cardiomyocytes after ischemia-reperfusion (I/R) are prevented by antioxidants such as N-acetyl L-cysteine (NAC), which is known to reduce oxidative stress by increasing the glutathione redox status, and N-(2-mercaptopropionyl)-glycine (MPG), which scavenges both peroxynitrite and hydroxyl radicals. For this purpose, isolated rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion, and cardiomyocytes were prepared to monitor changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Marked depression in the left ventricular developed pressure and elevation in the left ventricular end-diastolic pressure in I/R hearts were attenuated by treatment with NAC or MPG. Cardiomyocytes obtained from I/R hearts showed an increase in the basal level of [Ca(2+)](i) as well as augmentation of the low Na(+)-induced increase in [Ca(2+)](i), with no change in the KCl-induced increase in [Ca(2+)](i). These I/R-induced alterations in Ca(2+) handling by cardiomyocytes were attenuated by treatment of hearts with NAC or MPG. Furthermore, reduction in the isoproterenol-, ATP-, ouabain-, and caffeine-induced increases in [Ca(2+)](i) in cardiomyocytes from I/R hearts were limited by treatment with NAC or MPG. The increases in the basal [Ca(2+)](i), unlike the KCl-induced increase in [Ca(2+)](i), were fully or partially prevented by both NAC and MPG upon exposing cardiomyocytes to hypoxia-reoxygenation, H(2)O(2), or a mixture of xanthine and xanthine oxidase. These results suggest that improvement in cardiac function of I/R hearts treated with NAC or MPG was associated with attenuation of changes in Ca(2+) handling by cardiomyocytes, and the results support the view that oxidative stress due to oxyradical generation and peroxynitrite formation plays an important role in the development of intracellular Ca(2+) overload in cardiomyocytes as a consequence of I/R injury.

Neuregulin β1 enhances peak glutamate-induced intracellular calcium levels through endoplasmic reticulum calcium release in cultured hippocampal neuronsThis article is one of a selection of papers published in a special issue celebrating the 125th anniversary of the Faculty of Medicine at the University of Manitoba.

Modulation of intracellular free calcium levels is the primary second messenger system of the neuronal glutamatergic system, playing a role in regulation of all major cellular processes. The protein neuregulin (NRG) beta1 acts as an extracellular signaling ligand in neurons, rapidly regulating currents through ionotropic glutamate receptors. The effect NRG may have on glutamate-induced changes in intracellular free calcium concentrations has not been examined, however. In this study, cultured embryonic rat hippocampal neurons were treated with NRGbeta1 to determine a possible effect on glutamate-induced intracellular calcium levels. Long-term (24 h), but not short-term (1 h), incubation with NRGbeta1 resulted in a significantly greater glutamate-mediated acute peak elevation of intracellular calcium levels than occurred in vehicle-treated neurons. Long-term NRGbeta1 incubation significantly enhanced calcium increase induced by specific stimulation of metabotropic glutamate receptors, but did not significantly alter the N-methyl D-aspartate (NMDA)- or KCl-induced calcium increase and paradoxically decreased the effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) treatment on intracellular calcium. Metabotropic glutamate receptors cause increased intracellular free calcium via release of calcium from intracellular stores; thus this system was examined in more detail. NRGbeta1 treatment significantly (greater than 2-fold) enhanced calcium release from endoplasmic reticulum stores after stimulation of ryanodine receptors with caffeine, but did not significantly increase calcium release from endoplasmic reticulum mediated by inositol trisphosphate (IP3) receptors. In addition, ryanodine receptor inhibition with ruthenium red prevented the glutamate-induced increase in intracellular calcium levels in NRGbeta1-treated neurons. These data show that long-term NRGbeta1 treatment can enhance glutamate-induced peak intracellular calcium levels through metabotropic glutamate receptor activation by increasing endoplasmic reticulum calcium release through ryanodine receptors.

Nicotine stimulation of the medulla increases blood flow of the common carotid artery in cats

Microinjection of nicotine or glutamate into the dorsal facial area (DFA) of the medulla increases blood flow of common carotid artery (CCA). Whether there is a causal relationship between these two events is not known. Various agonists and antagonists for the nicotinic and glutamatergic receptors were microinjected through a four-barrel tubing into the DFA of anesthetized cats. Microinjections of nicotine [a non-selective nicotinic acetylcholine receptor (nAChR) agonist], choline (a selective α7-nAChR agonist), glutamate or KCl induced a modest increase in CCA blood flow. The nicotine- and choline-induced increases were reduced by α-bungarotoxin (an α7-nAChR antagonist) as well as MK-801 (a non-competitive NMDA receptor antagonist) or glutamate diethylester (a competitive AMPA/kainate receptor antagonist). The glutamate or KCl-induced increases were blocked by MK-801 and glutamate diethylester, but not by α-bungarotoxin. In conclusion, activation of nAChRs primarily via α7-nAChR caused a release of glutamate, which in turn activated NMDA and AMPA receptors, while cholinergic substance was not released into the DFA to activate the nicotinic receptor.

The Mechanisms of the Direct Action of Etomidate on Vascular Reactivity in Rat Mesenteric Resistance Arteries

Etomidate minimally influences hemodynamics at a standard induction dose in young healthy patients, but can cause significant systemic hypotension at higher doses for induction or electroencephalographic burst suppression (i.e., cerebral protection) in patients with advanced age or heart disease, and during cardiopulmonary bypass. However, less is known about its action on systemic resistance arteries. Using an isometric force recording method and fura-2-fluorometry, we investigated the action of etomidate on vascular reactivity in small mesenteric arteries from young (7-8 wk old, n = 179) and aged (96-98 wk old, n = 10) rats. In the endothelium-intact strips from young rats, etomidate enhanced the contractile response to norepinephrine or KCl (40 mM) at 3 microM but inhibited it at higher concentrations (>or=10 microM). The enhancement was still observed after treatment with N(G)-nitro l-arginine, tetraethylammonium, diclofenac, nordihydroguaiaretic acid, losartan, ketanserin, BQ-123, or BQ-788, but was not observed in aged rats. In the endothelium-denuded strips from young rats, etomidate (>or=10 microM) consistently inhibited the contractile response to norepinephrine or KCl without enhancement at 3 microM. In the fura-2-loaded, endothelium-denuded strips from young rats, etomidate inhibited norepinephrine- or KCl-induced increases in both intracellular Ca(2+) concentration ([Ca(2+)]i) and force. Etomidate still inhibited the norepinephrine-induced increase in [Ca(2+)]i after depletion of the intracellular Ca(2+) stores by ryanodine, which was sensitive to nifedipine. Etomidate had little effect on norepinephrine- or caffeine-induced Ca(2+) release from the intracellular stores or Ca(2+) uptake into the intracellular stores. During stimulation with norepinephrine or KCl, etomidate had little effect on the [Ca(2+)]i-force relation at low concentrations (<or=30 microM) but caused its downward shift at 100 microM. In small mesenteric arteries, etomidate influences the contractile response to norepinephrine or membrane depolarization through endothelium-dependent enhancing and endothelium-independent inhibitory actions. The enhancement is at least in part independent of nitric oxide, endothelium-derived hyperpolarizing factor, cyclooxygenase products, lipoxygenase products, angiotensin II, serotonin, or endothelin-1, but may involve some signaling pathway that is impaired by aging. The endothelium-independent inhibition is due to decreases in both the [Ca(2+)]i and myofilament Ca(2+) sensitivity in vascular smooth muscle cells. The decrease in [Ca(2+)]i would be due mainly to inhibition of voltage-gated Ca(2+) influx. The observed inability of lower concentrations (1-3 microM) of etomidate to cause significant vasodilation is consistent with minimal changes in hemodynamics during induction of anesthesia with etomidate in young subjects, whereas the observed vasodilator action of higher concentrations of etomidate might underlie systemic hypotension caused by higher doses of etomidate in the clinical setting.

Role of Protein Kinase Cζ and Calcium Entry in KCl-Induced Vascular Smooth Muscle Calcium Sensitization and Feedback Control of Cellular Calcium Levels

The degree of tonic force (F) maintenance induced in vascular smooth muscle upon K(+) depolarization with 110 mM KCl can be greatly reduced by inhibition of rhoA kinase (ROCK). We explored the possibility that a protein kinase C (PKC) isotype may also play a role in causing KCl-induced Ca(2+) sensitization. In isometric rings of rabbit artery, the PKC inhibitors, Go-6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), GF-109203X (2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide), and a cell-permeable (myristoylated) pseudosubstrate inhibitor of PKCzeta (PI(PKCzeta)) inhibited KCl-induced tonic F. A myristoylated pseudosubstrate inhibitor of PKCalpha/beta that inhibited phorbol dibutyrate-induced F slightly potentiated KCl-induced tonic F and attenuated 30 mM KCl-induced F. Although the ROCK inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)-sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride], reduced basal phosphorylation of myosin light-chain phosphatase-targeting subunit at Thr853 (MYPT1-pT853), 3 and 10 muM GF-109203X inhibited only KCl-stimulated phosphorylation, not basal MYPT1-pT853. In fura-2-loaded tissues, GF-109203X and PI(PKCzeta) elevated basal [Ca(2+)](i) (calcium) and potentiated KCl-induced tonic increases in calcium while reducing KCl-induced tonic increases in F. Blockade by nifedipine of Ca(2+) entry through voltage-operated Ca(2+) channels reduced KCl-induced Ca(2+) sensitization and KCl-stimulated but not basal MYPT1-pT853. These data together support a model in which ROCK and PKCzeta are constitutively active and function in "resting" muscle to regulate the basal levels of MYPT1-pT853 and calcium, respectively. In this model, KCl-induced increases in calcium activate PKCzeta to feed forward and cause additional MYPT1-pT853 above that induced by constitutive ROCK, permitting Ca(2+) sensitization and strong F maintenance. Active PKCzeta also feeds back to attenuate the degree of KCl-induced increases in calcium.

Activity‐dependent regulation of tyrosine hydroxylase expression in the enteric nervous system

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH-IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH-IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 microm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP-IR but not nNOS-IR enteric neurons. The KCl-induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 microm), of noradrenaline (1 microm) and of the alpha(2)-adrenoreceptor agonist clonidine (1 microm). Combining pharmacological and calcium imaging studies, we have further shown that L-type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal-regulated kinases were required for the increase in the proportion of TH-IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.