Blockade of L-type Ca2+ channels suppresses activation of calcineurin and development of right ventricle cardiac hypertrophy induced by chronic hypoxia

AIM: To study the role of calcineurin in the progression of right ventricle cardiac hypertrophy in the chronic hypoxia rats and examine the effect of Ca²⁺ channel blockers on the activation of calcineurin. METHODS: Sixty rats were divided into three groups: treatment group with amlodipine besylate ablets, chronic hypoxia group, normal control group with normal oxygen. The rats in treatment group and chronic hypoxia group were exposed to normobaric chronic hypoxia(10±0.5)% O₂ for 21 days. All hearts were removed immediately after dissection for further investigation. RESULTS: (1)The RV/(LV+S),RV/BW were significantly higher in hypoxia group than that of control group and treatment group(P<0.01,respectively); (2) Right ventricular cardiomyocytes [Ca²⁺]_i in treatment group were significantly higher than that of control group and lower that that of hypoxia group(P<0.01,respectively); (3) The activity of calcineurin of the heart in hypoxia group were significantly increased when compared with control group. Amlodipine besylate ablets apparently suppressed the activity of calcineurin(P<0.01). CONCLUSION: Calcineurin possibly plays a role in the progression of right ventricle cardiac hypertrophy in the chronic hypoxia rats;Blockade of L-type Ca²⁺ channels with amlodipine besylate ablets effectively prevents its development possibly by inhibition of calcium inflow and suppression of the calcineurin activity.     

Role of potassium channels in the regulation of intracellular free Ca2+ concentration of pulmonary artery smooth muscle cells in rats

AIM: To investigate the role of potassium channels in the regulation of intracellular free calcium concentration ( [Ca²⁺]_i) of pulmonary artery smooth muscle cells (PASMCs) in rats. METHODS: The fluorescence Ca²⁺ indicator Fura-2/AM was used to observe [Ca²⁺]_i of rat PASMCs in normal and chronic hypoxic condition. The influences of potassium channels on PASMCs proliferation were assessed by MTT assay. RESULTS: 1. In normoxic condition, [Ca²⁺]_i was (156.91±8.60) nmol/L, and in hypoxic condition, [Ca²⁺]_i was (294.01±16.81) nmol/L. 2. In normoxic condition, the voltage-dependent K⁺-channel antagonist 4-aminopyridine (4AP), but not the Ca²⁺-activated K⁺-channel antagonist tetraethylammonium (TEA) and the ATP-sensitive K⁺-channel antagonist glibenclamide (Glib) increased [Ca²⁺]_i. 3. In hypoxic condition, 4AP and TEA caused the rise in [Ca²⁺]_i , but Glib had no effect on [Ca²⁺]_i. 4. MTT assay showed that 4AP increased the value of absorbing light degree (A value) in normoxic and hypoxic condition (0.582±0.062,0.873±0.043,respectively, P＜0.01), TEA increased A value only in hypoxic condition, and Glib had no effect on the proliferation of PASMCs. CONCLUSIONS: K_V plays an important role in the regulation of [Ca²⁺]_i and proliferation of PASMCs. K_Ca serves as distinct responsive roles in the regulation of proliferation of PASMCs in hypoxic condition. K_ATP has no effect on [Ca²⁺]_i and proliferation of PASMCs in normoxic and hypoxic conditions.     

Change of L-type voltage-gated Ca2+ channel current during focal brain ischemia in diabetic rats

AIM: To observe the change of L-type voltage-gated Ca²⁺ channel current during focal brain ischemia in the normal rats and the rats with diabetes mellitus (DM). METHODS: Combination of high-fat diet with streptozotocin (STZ) was used to establish DM animal model. The operation of middle cerebral artery occlusion (MCAO) with monofilament on the rats was performed. The animals were divided into sham operation group, MCAO 1 h group, MCAO 3 h group, MCAO 6 h group, MCAO 24 h group, DM sham operation group, DM+MCAO 1 h group, DM+MCAO 3 h group, DM+MCAO 6 h group and DM+MCAO 24 h group. The score of neural function was determined to judge the degree of palsy in the rats in MCAO 24 h group and DM+MCAO 24 h group. The changes of L-type voltage-gated Ca²⁺ channel current of cortex neurons during ischemia were measured using the whole-cell patch clamp technique. RESULTS: The rats in DM+MCAO 24 h group awaked slowly, and the degree of semiplegia was more serious than that in the rats in MCAO 24 h group. The score of neural function in DM+MCAO group was higher than that in MCAO group (P<0.05). The longer the ischemic time was, the higher L-type voltage-gated Ca²⁺ channel current was observed in MCAO group and DM+MCAO group (P<0.05). L-type voltage-gated Ca²⁺ channel current in DM+MCAO group was higher than that in MCAO group at each time point(P<0.05). CONCLUSION: The aggratation of ischemic injury during DM+MCAO is probably associated with Ca²⁺ overload induced by calcium channel opening and current increasing.     

Role of K+ channels in hypoxic pulmonary vasoconstriction in normal and chronic hypoxic rats

AIM:To investigate the role of K⁺ channels in the decreased hypoxic pulmonary vasoconstriction(HPV) in chronic hypoxic rats. METHODS:Blockers of three kinds of K⁺ channels, 4-AP(voltage dependent K⁺ channel blocker), TEA(Ca²⁺ activated K⁺ channel blocker), GLIB(ATP sensitive K⁺ channel blocker) were used in isolated perfused rat lungs to detect the role of K⁺ channels in HPV. RESULTS:In normal rats, 4-AP and TEA, but not GLIB, both elicited a significant increase in pulmonary artery baseline pressure, and also potentiated the acute hypoxic pulmonary vasoconstriction. In chronic hypoxic rats, the HPV is significantly decreased, while 4-AP, TEA, GLIB all elicited a significant but smaller increase in pulmonary artery baseline pressure. Additionally, all these three blockers potentiated the HPV stronger in chronic hypoxic rats than in control rats. CONCLUSION:The opening of Kv, K_Ca, K_ATP might modulate the hypoxic pulmonary vasoconstriction in isolated rat lungs, and the increase in this modulation by potassium channel in chronic hypoxic rats might play a role in its decrease in HPV.     

Salvia miltiorrhiza antagonized the alterations of contraction and intracellular calcium of rat ventricular cardiomyocytes induced by anoxia and reoxygenation

AIM:To study the effect of salvia miltiorrhiza (SM) on cell contraction and intracellular calcium of enzymatically isolated rat ventricular myocytes during normoxia and anoxia/reoxygenation.METHODS:Contraction and intracel ular calcium were determined with video tracking system and spectrofluorometric method,and the chemical anoxic method was employed. RESULTS:The ±dL/dt_max, dL of cell contraction and the amplitude of [Ca²⁺]_i in the cardiomyocytes following SM treatment were decreased in a dose-dependent manner. During anoxia, the ±dL/dt_max, dL and amplitude of [Ca²⁺]_i were decreased, while the diastolic Ca²⁺ level was elevated compared with control group. All the contractile parameters and the diastolic Ca²⁺ level were back toward pretreatment values during reoxygenation, but could not return to control level. After the treatment with SM (3 g/L), ±dL/dt_max and dL of cell contraction and the amplitude of [Ca²⁺]_i were higher and the diastolic Ca²⁺ level was lower than those in anoxia/reoxygenation group. CONCLUSION:SM antagonized effects of anoxia and reoxygenation on cell contraction and intracellular calcium in isolated ventricular myocytes.     

Hypoxia facilitates rapid pacing-induced calcium transient alternations in ventricular myocytes

AIM:To explore the effect of hypoxia on rapid pacing-induced calcium transient alternations in ventricular myocytes.METHODS:Ventricular myocytes were isolated from the heart of adult SD rats and cultured in serum-free hypoxic fluid to set up an in vitro model of hypoxia-induced cardiac injury. The calcium transient and its alternations were investigated under confocal laser scanning microscope. The mitochondrial function was also examined by WST-8 kit. RESULTS:Under normoxic condition, the ventricular myocytes were claviform. Low frequency of pacing, ranging from 60 to 240 min^-1, induced calcium transient, but not calcium transient alternations, which was elicited by the pacing over a threshold frequency of(288 ±27)min^-1. Exposure of the ventricular myocytes to hypoxia did not obviously affect the morphology of the cells, but reduced the threshold frequency of pacing to(227±26) min^-1(P<0.05). Additionally, exposure of the cells to hypoxia repressed the activity of mitochondrial dehydrogenase from(100.2±8.7)%(control group) to(57.6±7.5)%, which was partially blocked by L-type Ca²⁺ channel inhibitor. CONCLUSION:Hypoxia facilitates calcium transient alternations induced by rapid pacing, and the calcium transient alternations are involved in the hypoxia-injured mitochondria function.     

Effect of interleukin-2 on intracellular calcium levels in rat ventricular myocytes during anoxia and reoxygenation

AIM: To investigate the effect of interleukin-2(IL-2) on the intracellular calcium in electrically stimulated adult rat ventricular myocytes during anoxia and reoxygenation. METHODS: The isolated cardiac ventricular myocytes were exposed to 5 min anoxia followed by 10 min reoxygenation. Chemical anoxia was introduced by Krebs-Henseleit(K-H) solution containing 10^-3 mol/L sodium dithionite. The spectrofluorometric method was used to verify intracellular calcium transient with fura-2/AM as calcium fluorescence probe. RESULTS: It was shown that during anoxia, the amplitude of Ca²⁺ transient was decreased, diastolic [Ca²⁺]_i, time to peak and time to relaxation of Ca²⁺ transient were increased. All the parameters were got back but did not returned to the pre-anoxia level during reoxygenation. IL-2 at 2×10⁵ U/L administrated during anoxia aggravated the effect of rexoxygenation on [Ca²⁺]_i transient. Pretreatment with a specific κ opioid antagonist, nor-BNI(10^-8 mol/L), abolished the effect induced by IL-2 during anoxia on the [Ca²⁺]_i transients, whereas specific δ opioid antagonist, naltrindole(10^-6 mol/L), did not cancel the effect. CONCLUSION: It is concluded that administration of IL-2 during anoxia aggravated the effect of reoxygenation on the [Ca²⁺]_i transients of isolated ventricular myocytes, which was mediated by cardiac κ opioid receptor pathway.     

Effect of Ba2+ concentration on L-type Ca2+ channel in hypothalamic neurons

AIM:To investigate the effect of Ba²⁺ concentration on L-type of Ca²⁺ channel in hypothalamic neurons.METHODS:The cell acute isolation technique and cell-attached patch-clamp technique were used.RESULTS:The slope conductance of L-type Ca²⁺ channel were 28.6 pS (110 mmol/L) and 19.1 pS (10 mmol/L), and the open probability (NP₀) obviously different with different Ba²⁺ concentration as carrier. CONCLUSION:Ba²⁺ concentration had the obvious effect on the L-type Ca²⁺ channel.     

Nuclear calcium oscillation in cultured neonatal rat myocytes

AIM: To determine whether nuclear Ca²⁺ is independently regulated from the cytosolic Ca²⁺ and nuclear Ca²⁺ oscillation induced by many modulating factors in cultured rat neonatal myocytes and its mechanism. METHODS: Rat neonatal cardiac myocytes were cultured, and fluo-4/AM was loaded as calcium probe. The changes of cytosolic and nuclear Ca²⁺ were observed by confocal laser microscopy. RESULTS: Calcium fluorescent intensity oscillated slightly in myocardiocytes and the average intensity was much higher in the nucleus than that in the cytosole. Ca²⁺ oscillation in nucleus and cytosole induced by norepinephrine, isoproperenol, ATP were completely blocked by Ca²⁺-ATPase antagonist thapsigargin (10^-6 mol/L),L-type Ca²⁺ channel blocker verapermil (500 μmol/L) and KCl (20 mmol/L). Ca²⁺-ATPase antagonist thapsigargin completely blocked the propagation of Ca²⁺ waves and simutaneouly induced a temporary Ca²⁺ increase followed by a magnificient drop and loss of response to norepinephrine. CONCLUSIONS: The results suggested that generation and maintenance of calcium oscillation both in cytosole and nucleus depended on extracellular Ca²⁺ influx, membrane potential, Ca²⁺ release and uptake of cytosolic and nuclear calcium stores. The difference between cytosolic calcium and nuclear calcium indicated that calcium regulating system relatively independent of cytosole may exist in nucleus.     

Relationship between thromboxane synthase gene and prostacyclin synthase gene expression and pulmonary hypertension induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on gene expression of thromboxane synthase and prostacyclin synthase in pulmonary arterioles. METHODS: Sprague-Dawley rats were randomly divided into two groups: control group and hypoxic hypercapnic group. TXS mRNA and PGI₂-SmRNA were observed in pulmonary arterioles by in situ hybridization. RESULTS: mPAP, weight ratio of right ventricle (RV) to left ventricle plus septum(LV+S), contents of TXB₂ and 6-keto-PGF1_α in plasma and lung and TXS mRNAin pulmonary arterioles were much higher in rats of hypoxic hypercapnic group than those of control group. Differences of PGI₂-SmRNA in pulmonary arterioles were not significant in two groups. Light microscopy showed hypertrophy of vessel smooth muscle cells and vessel cavity straitness were found in hypoxic hypercapnic group. CONCLUSION: Changes of gene expressions of thromboxane synthase and prostacyclin synthase and imbalance of TXA₂/PGI₂ may play an important role in hypoxic hypercapnic pulmonary hypertension.