Arginine Vasopressin Enhances The T-type Calcium Current Through Intracellular Ca²⁺-and PKC-dependent Signaling Pathway

Objective: Arginine vasopressin?AVP?is a nanopeptide neurohypophysis hormone synthesized in the hypothalamic supraoptic nucleus and hypothalamic paraventricular nucleus.AVP has be used for cardiac arrest and the recovery of low vascular reactivity after severe trauma shock as a potent vasoconstrictor,for maintaining hemodynamic stability.And AVP V2 receptor antagonist drugs have also been used to treat hyponatremia for relieving heart failure symptoms Because the it has high selectivity,it can eliminate warter not sodium,increases the exclusion of free water and had no significant effect of electrolyte.AVP will contribution the cardiac fibroblasts to stronger muscle fibroblasts,cause myocardial fibrosis by V1 a receptor.Many studies have confirmed the AVP can cause arrhythmia by accommodate L type calcium channels,KCNQ potassium channels,in a previous study Our laboratory has proved in acute and chronic condition AVP can increase T type calcium currents,but the mechanism has not understood yet.In the present study,we use the whole-cell patch-clamp technique to research the mechanism.from the calcium sensitivity adjustment molecular PKC pathway.Main contents include three parts:?1?To further research the effect of AVP to ICa.T in neonatal rat myocardial cells.?2?The relationship between effect of AVP to ICa.T and intracellular different calcium concentration.?3?The mechanism of AVP enhancement T type calcium current via PKC signal pathways.Methods:1 Neonatal rat cardiomyocytes: preparations and cultureNeonatal cardiomyocytes were prepared from 1–3-day-old Sprague Dawley rats.The cardiomyocytes were plated onto 35 mm culture dishes and cultured in Dulbecco's modified Eagle's medium,supplemented with 10% fetal bovine serum at 37°C under 5%CO2/95%O2.for 24–48 h before electrophysiological measurements.2 Whole-cell current recordingsMacroscopic L-type Ca²⁺ channel currents?ICa.L?and T-type Ca²⁺ channel currents?ICa.T?were recorded at room temperature?20–23°C?by voltage-clamp technique.the corresponding bath solution is?millimoles?:?TEA?-Cl 136,Ca Cl2 2,Mg Cl2 2,HEPES 25,glucose 20?p H was adjusted to 7.3 with TEA-OH?.and five different pipette solutions were prepared by means of different Ca²⁺ concentrations according to the equation by Fabiato [15] together with?in m M?Cs Cl 130,Mg Cl2 2,ATP-Mg 2,GTP-Na 0.5 and HEPES 5,where p H was adjusted to 7.2 with 1N Cs OH?solutions A,B,C,D and F in Table 1?.L-type plus T-type calcium current: The holding potential is-100 m V,and the test potential is from-90 m V to +50m V in 10 mv step,step width 300 ms.L-type calcium current:The holding potential is-50 m V,and the test potential is from-40 m V to +50m V in 10 mv step,step width 300 ms.Effects of AVP?300n M?on Sprague Dawley rats cardiac T-type Ca²⁺ channel current.?1?It was measured through contract the current before using drug and the current after using drug in a cell,and in the control condition and in AVP?300 ?M?application in 5 min recorded in [Ca²⁺]i condition?p Ca=7 p Ca=8 p Ca=9 p Ca=10 p Ca=11?.T-type calcium current was recorded before using Drug and after using drug in a cell.?2?To further investigate the underlying modulatory mechanism of AVP on T-type Ca²⁺ channels,we used protein kinases inhibitor and V1 a inhibitor culture cell for 1h and recorded T-type calcium current before using AVP and after using AVP in a cell for 5min.in p Ca=7.2.3 Data acquisition and statistical analysisThe data were acquired by using computer software?Pulse/Pulse Fit,V.10,HEKA Germany?.The group data are shown as means±SD.Analysis of variance and multiple comparisons were used for the different [Ca²⁺]i condition.,and Student's t test was used for the comparison of two groups before and after using AVP.Differences were considered significant when p values were less than 0.05.Results:1 T-type Ca²⁺ channel currents change by AVP was assessed by different p Ca condition: AVP increases T type calcium current.effect of AVP on ICa.T is highly dependent on [Ca²⁺]i.300n M AVP acute group and the control group in p Ca=11[Ca²⁺]i condition,the increase rate of the current is?9.63±0.83?%?n=12,P<0.05⁰.01?.300 n M AVP acute group and the control group in p Ca=10 [Ca²⁺]i condition,the increase rate of the current is?27.83±2.43?%?n=12,P<0.01?.300 n M AVP acute group and the control group in p Ca=9 [Ca²⁺]i condition,the increase rate of current is?33.28±4.90?%?n=13,P<=0.001?.300 n M AVP acute group and the control group in p Ca=8 [Ca²⁺]i condition,the increase rate of the current is?37.30±3.36?%?n=10,P<0.05?.300 n M AVP acute group and the control group in p Ca=7 [Ca²⁺]i condition,the increase rate of the current is?39.75±4.41?%?n=12,P<0.05⁰.01?.when the calcium ion concentration in the cell is p Ca7,T type calcium channel current increased significantly,and when the calcium ion concentration in the cell is p Ca=11,T calcium current did not increase apparently,when we analysis the data of the increase rate in condition of p Ca = 7 and p Ca = 11 P < = 0.001,these data suggest that with the increase of intracellular calcium ion concentration the effect of AVP on T calcium current are more apparent.AVP regulation function on T type calcium current depends on the change of calcium ion concentration in the cell.2 AVP can increase T type calcium currents by PKC signal pathwaysTo further explore the mechanism of action of AVP of ICa T,We use protein kinase inhibitors to culture cell for 1 hour and record the T type current before and after we use 300 n M AVP in containing the physiological state of calcium ion concentration p Ca = 7.2.The control group of 300 n M AVP,the ICa.T increases 38.67±8.74%.The actions of protein kinases were inhibited by H-89?protein kinase A,PKA inhibitor 10?mol/L?,ICa.T was increased by 300 n M AVP by 36.06±7.18%.inhibited by chelerythrine?PKC inhibitor 5?mol/L?,ICa.T was increased by 300 n M AVP by inhibited by 14.68±5.21%.KT5823?protein kinase G,PKG inhibitor 1?mol/L?,ICa.T was increased by 300 n M AVP by 37.87±6.36%.KN-62?calmodulin kinase II inhibitor 3?mol/L?,ICa.T was increased by 300 n M AVP by 39.9±7.26% or PD 98059?extracellular signal-regulated kinase inhibitor 10?mol/L?,ICa.T was increased by 300 n M AVP by 37.60±5.36%.We analysis the increase rate of the experimental group and control group,the experimental group compared with control group respectively.PKC inhibitor and the V1 a receptor inhibitor group P < 0.05.Other group P values were greater than 0.05,the results show that adding PKC inhibitors the effect of AVP to T type calcium current weaken,namely AVP can increase T type calcium currents by PKC signal pathways.Conclusions:Intracellular Ca²⁺-and PKC-dependent upregulation of T-type Ca²⁺ channels in AVP-stimulated cardiomyocytes.