| Background:Ca2+ is one of the most strictly regulated ions in the cell, because it is involved in many important signal cascades by activating various Ca2+-binding proteins, such as kinases, phosphatases, and proteases. Ca2+ signals induce various physiological responses such as muscle contraction, vesicle secretion, cell differentiation, proliferation, and cell death. Whereas some of the responses to the Ca2+ signal are cell-type specific, multiple Ca2+ signals are used for different responses in the same cell. In cardiac myocytes, for example, Ca2+ triggers muscle contraction in response to every action potential, whereas in pathophysiological circumstances (e.g. cardiac hypertrophy), the Ca2+ signal induces phenotypic remodeling of the myocytes via gene expression by activating transcription factors. Moreover, the prolonged Ca2+ signal leads to cell death. It is thus important to clarify how different Ca2+ signal systems can be set up to control different cellular functions for the better understanding of the Ca2+ signal transduction in native tissues. Ca2+ used for cell signaling is derived either from the extracellular fluid or internal stores. In the former case, many kinds of plasma membrane depolarization, extracellular and intracellular ligands, and mechanical stretch. In the latter case, ryanodine receptor (RyR) and inositol-1,4,5-triphosphate (InsP3) receptor are responsible for the release of Ca2+ from the internal Ca2+ store such as the endoplasmic reticulum (ER) or its muscle equivalent, the sarcoplasmic reticulum (SR). The excitation of cells (action potential) was connected with the contraction of cells through Ca2+ which is called excitation-contraction coupling, ECC. Ca2+ influx through the L-type Ca2+ channels which were caused open by the excitation of myocyte result the sarcoplasmic reticulum Ca2+ channels open, a large of Ca2+ release in the form of Ca2+-induced Ca2+ release. The concentration of Ca2+ intracytoplasm burst at a short time which is called calcium transients. Calcium transients cause the cell contracting through the troponin, which is the most obvious, stronger and typical calcium signal change intracellular. We can understand the function, activity, characteristic and state of the myocytes by observing the calcium transient. So we record the calcium transients of rat ventricular myocytes which were induced by local field stimulation in different drug circumstance with the help of confocal system Zeiss LSM510 META. Discussing the mechanism and hope it can provide some theory for the cardiovascular disease therapy.Objective:(1) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different hyperkalemia, analysising the information in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different hyperkalemia.(2) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different hypokalemia, analysising the information in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different hypokalemia.(3) Observing the changes of calcium signals about the ventricular myocytes of adult SD rats in different concentration of nifedipine, analysising information the in the images and discussing the possible mechanism which affect the calcium signals of the ventricular myocytes about adult SD rats in different concentration of nifedipine.Materials and methods:Adult Sprague-Dawley rats of either sex (weight, 200-350g,) were anesthetized with sodium pentobarbital (50 mg/kg IP). Once anesthetized, the heart was rapidly excised and mounted on a Langendorff perfusion apparatus. Ventricular myocytes were dispersed by enzymatic (0.6mg/ml collagenase; typeⅡ+0.06mg/ml protease+1mg/ml BSA) digestion. Enzymatically isolated ventricular myocytes were loaded with Ca2+ indicator Fluo-4-AM (15μmol/L) for 15 min, followed by a 10 min rest allowing for deesterfication of the indicator. The criteria for cell selection included rod shape, clear striation and clean cell surface, and lack of spontaneous contractions during a 1-min observation period. Ca2+ images were acquired by using a Zeiss LSM510 confocal microscopy. Computer programs for the calcium transients were coded in Interactive Data Language (IDL, Research Systems, Boulder, CO). Calcium transients were measured as△R =△F/F0 , where F refers to the present Fluo signal intensity, F0 the background Fluo signal intensity, and△F/F0 the alteration of F/F0. Results:1. The changes of calcium signals on the circumstance of hyperkalemia: (1) the peak of calcium transients is 7.56±0.56 (△F/F0) at the condition of 5.4mmol/L KCl, line scanning. The peak of calcium transients is 7.90±0.63 (△F/F0) after perfusing with 7.5mmol/LKCl. The value of normalization is 1.07±0.03>1, the peak of calcium transients become higher than the control; The peak of calcium transients become lower step by step after perfusing with 10,20,50 mmol/L KCl (P<0.05). (2) The background calcium signals become higher step by step after perfusing with 10,20,50mmol/L KCl (P<0.05).The background calcium signals is triplicity than the control, the calcium homeostasis was broken intracellular ventricular myocytes.2. The changes of calcium signals on the circumstance of hypokalemia:(1) the peak of calcium transients (6.85±0.70) which induced from 5 ventricular myocytes (observing 6 ventricular myocytes) after perfusing with 2mmol/L KCl is not significant, compared with the control (6.54±0.21) (P>0.05); FWHM (meaning the time course of calcium release) is 203.31±17.12 ms on the circumstance of 2 mmol/L KCl, which is significant compared with the control 176.46±17.08 ms (P<0.05). The peak of calcium transients reduce slightly comparing with the control (P>0.05), FWHM 222.49±17.00 ms prolonged obviously than the control (P<0.05) after perfusing with the normal extracellular fluid. It indicated that 2mmol/L KCl can induce the prolongation of the time course about calcium transients, and the effects can not recover at a short time. We observed a ventricular myocyte can induced calcium transients at first, then have no reaction to the local stimulation and appear calcium transients in the form of spontaneous.(2) We observed 9 ventricular myocytes at 1 mmol/L KCl, 5 cells can induce calcium transients, the peak increase but P>0.05. FWHM 209.0±22.1 ms after washing out 1 mmol/L KCl with normal extracellular fluid prolonged significantly compared with the control 178.4±18.8 ms (P<0.05). 1 cell can induce calcium transients (the time course prolonged), then has no reaction to the local stimulation, then appear calcium transients in the form of spontaneous: 1 cell has no reaction to the local stimulation at first, then can induce calium transients, then has no reaction to the local stimulation; 2 cells have no reaction to the local stimulation, appearing calcium transients in the form of spontaneous.(3) We observed 8 ventricular myocytes at the circumstance of 0 mmol/L KCl. 7 cells appear calcium transients in the form of spontaneous, then the induce calcium transients recover after perfusing with normal extracellular fluid but the time course 211.0±9.7 ms prolong than the control 155.4±3.8 ms; 3 cells can induce the calcium transients to the local stimulation at first (the time course prolong), then appear calcium transients or calcium wave in the form of spontaneous; 3 cells have no reaction to the local stimulaton then appear calcium transients or calcium wave in the form of spontaneous; 1 cell appear calcium transients or calcium wave in the form of spontaneous;1 cell calcium transients reduce quickly, then recover, but the time course prolong, the cell do not contract.3. The changes of calcium signals on the different concentration of nifedipine: The peak of calcium transients reduced at the different concentration of nifedipine. The value of normalization on the 5μmol/L nifedipine is the lowest, the effect is better than the 2μmol/L nifedipine (P<0.05). The background calcium signals haves no obvious change on the different concentrations of nifedipine.Conclusions:1. Hyperkalemia can break the homeostasis of intracellular calcium about ventricular myocytes, the peak of calcium transients increase at first then reduce with the [K+]o increasing.The depression about the peak of calcium transients has correlated to the increase about the background calcium signals closely when [K+]o increasing (10,20,50mmol/L).2. The different cells have different reaction to the hypokalemia, but the tendency is that hypokalemia can disturb the homeostasis of intracellular calcium about ventricular myocytes. The reduce slightly of [K+]o cause the change of calcium transients step by step (the peak do not change , the time course of calcium release prolong), the 0mmol/L KCl cause the calcium signals of ventricular myocytes jumping change, we can observe the calcium transients or calcium waves in the form of spontaneous.3. Nifedipine can reduce the magnitude of calcium transients, but have no effects on the background calium signals about ventricular myocytes.
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