Real-time detection of α1A-AR movement stimulated by phenylephrine in single living cells

Aim： To investigate the movement of α1A-adrenergic receptors（α1A-AR） stimulated by agonist, phenylephrine （PE）, and the dynamics of receptor movement in real time in single living cells with millisecond resolution. Methods： We labeled α1A-AR using the monoclonal, anti-FLAG （a kind of tag） antibody and Cy3-conju- gated goat anti-mouse IgG and recorded the trajectory of their transport process in living HEK293A cells stimulated by agonist, PE, and then analyzed their dynamic properties. Results： The specific detection of α1A-AR on the surface of living HEK293A-α1A cells was achieved, α1A-AR internalize under the stimulation of PE. After the cells were stimulated with PE for 20min, apparent colocalization was found between α1A-AR and F-actins. After 40 rain stimulation of PE, trajectories of approximate linear motion in HEK293A-α1A cells were recorded, and their velocity was calculated. Conclusion： The specific labeling method on the living cell surface provides a convenient means of real-time detection of the behavior of surface receptors. By this method we were able to specifically detect α1A-AR and record the behavior of individual particles of receptors with 50 ms exposure time in real time in single living cells.     

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardio- myocytes

β-adrenergic receptor （β-AR） agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 （Cx43）, the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication （GJIC） function.
Methods： The level of Cx43 expression in neonatal rat cardiomyocytes （NRCM） was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay.
Results： Stimulation of β-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective β2-AR inhibitor ICI 118551, but not β1-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and Gi protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function.
Conclusion： Taken together, β2-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A-and G1-dependent manner.     

Blockade of L-type calcium channel in myocardium and calcium-induced contractions of vascular smooth muscle by CPU 86017

AIM: To assess the blockade by CPU 86017 on the L-type calcium channels in the myocardium and on the Ca^2 -related contractions of vascular smooth muscle. METHODS: The whole-cell patch-clamp was applied to investigate the blocking effect of CPU 86017 on the L-type calcium current in isolated guinea pig myocytes and contractions by KCl or phenylephrine (Phe) of the isolated rat tail arteries were measured. RESULTS: Suppression of the L-type current of the isolated myocytes by CPU 86017 was moderate, in time- and concentration-dependent manner and with no influence on the activation and inactivation curves. The IC50 was 11.5 μmol/L. Suppressive effect of CPU 86017 on vaso-contractions induced by KCl 100 mmol/L, phenylephrine 1 μmol/Lin KH solution (phase 1),Ca^2 free KH solution ( phase 2), and by addition of CaCl2 into Ca^2 -free KH solution (phase 3) were observed. The IC50 to suppress vaso-contractions by calcium entry via the receptor operated channel (ROC) and Voltage-dependent channel (VDC) was 0.324μmol/L and 16.3μmol/L, respectively. The relative potency of CPU 86017 to suppress vascular tone by Ca^2 entry through ROC and VDC is 1/187 of prazosin and 1/37 of verapamil, respectively.CONCLUSION: The blocking effects of CPU 86017 on the L-type calcium channel of myocardium and vessel are moderate and non-selective. CPU 86017 is approximately 50 times more potent in inhibiting ROC than VDC.     

Isoform-specific regulation of the Na^＋-K^＋ pump by adenosine in guinea pig ventricular myocytes

Aim The present study investigated the effect of adenosine on Na^＋-K^＋ pumps in acutely isolated guinea pig （Cavia sp.） ven- tricular myocytes. Methods： The whole-cell, patch-clamp technique was used to record the Na^＋-K^＋ pump current （Ip） in acutely isolated guinea pig ventricular myocytes. Results- Adenosine inhibited the high DHO-affinity pump current （Ih） in a concentration-dependent manner, which was blocked by the selective adenosine A1 receptor antagonist DPCPX and the general protein kinase C （PKC） antagonists staurosporine, GF 109203X or the specific 6 isoform antagonist rottlerin. In addition, the inhibitory action of adenosine was mimicked by a selective A1 receptor agonist CCPA and a specific activator peptide of PKC-5, PPl14. In contrast, the selective A2A receptor agonist CGS21680 and A3 receptor agonist CMB-MECA did not affect Ih. Application of the selective A2A receptor antagonist SCH58261 and A3 receptor antagonist MRSl191 also failed to block the effect of adenosine. Furthermore, H89, a selective protein kinase A （PKA） antagonist, did not exert any effect on adenosine-induced Ih inhibition. Conclusion： The present study provides the electrophysiological evidence that adenosine can induce significant inhibition of Ih via adenosine A1 receptors and the PKC-6 isoform.     

Inhibitory effect of ginsenoside Rb1 on calcineurin signal pathway in cardiomyocyte hypertrophy induced by prostaglandin F2α

Aim： To examine the antihypertrophic effect of ginsenoside Rb1 （Rb1） induced by prostaglandin F2α （PGF2α） in vitro and to investigate the possible mechanisms involved in the calcineurin （CAN） signal transduction pathway. Methods： The cardiomyocyte hypertrophy induced by PGF2α and the antihypertrophic effect of Rb1 were evaluated in primary culture by measuring the cell diameter, protein content, and atrial natriuretic peptide （ANP） mRNA expression. ANP and CaN mRNA expressions, CaN and its downstream effectors NFAT3 and GATA4 protein expressions, and the intracellular free Ca^2＋ concentration （[Ca^2＋]i） were assayed by RT-PCR, Western blot, and fluorescent determination using Fura 2/AM, respectively. Results： PGF2α （100 nmol/L） significantly increased the cardiomyocyte diameter, protein content and [Ca^2＋]i, and promoted ANP, CaN mRNA, and CaN/NFAT3/GATA4 protein expressions, which were inhibited by either Rb1 in a concentration-dependent manner （50, 100, and 200 μg/mL） or L-arginine （1 mmol/L）. N^G-nitro-L-arginine-methyl ester, a nitric oxide synthase inhibitor, could abolish the effects of L-arginine, but failed to change the effects of Rb1 in the experiments above. Conclusion： The present data implicate that Rb1 attenuates cardiac hypertrophy, the underlying mechanism may be involved in the inhibition of the Ca^2＋-CaN signal transduction pathway.     

Tetrandrine and related bis—benzylisoquinoline alkaloids from medicinal herbs：cardiovascular effects and mechanisms of action

Tetrandrine(TET),a bis-benzylisoquinoline alkaloid purified and identified an active ingredient in a Chinese medicinal herb,Radix Stephanae tetrandrae,has been used traditionally for the treatment of congestive circulatory disorder and inflammatory diseases.TET,together with a few of its structural analogues,has long been demonstrated to have antihypertensive action in clinical as well as animal studies.Presumably,the primary anti-hypertensive action of TET is due to its vasodilatory properties.TET prevents or inhibits vascular contraction induced by membrane depolarization with KCl or α-adrenoceptor activation with phenylephrine (PE).TET(30μmol/L) also inhibits the release of endothelium-derived nitric oxide(NO) as well as NO production by inducible NO synthase.TET apparently inhibits multiple Ca^2 entry pathways as demonstrated in cell types lacking the L-type Ca^2 channels.In cardiac muscle cells,TET inhibits both L-and T-type Ca^2 channels.In addition to its actions on cardiovascular tissues,TET may also exert its anti-hypertensive action via a Ca^2 -dependent manner on other tissues intimately involved in the modulation of blood pressure control,such as adrenal grands.In adrenal glomerulosa cells,KCl-or angiotensin II-induced aldosterone synthesis is highly dependent on extracellular Ca^2 .Steroidogenesis and Ca^2 -influx in bovine adrenal glomerulosa cells have been shown to be potently inhibited by TET.In bovine adrenal chromaffin cells,TET inhibits Ca^2 currents via L-and N-type channels as well as other unidentified channels with IC50 of 10μmol/L.Other than the Ca^2 antagonistic effects.TET also interacts with the α-adrenergic receptors and muscarinic receptors based on functional as well as radioligand binding studies.Apart from its functional effects,TET and related compounds also exert effects on tissue structures,such as remodelling of hypertrophied heart and inhibition of angiogenesis,probably by causing apoptotic responses.TET is also known for its anti-inflammatory and anti-fibrogenic actions,which make TET and related compound potentially useful in the treatment of lung silicosis,liver cirrhosis,and rheumatoid arthritis.     

Crosstalk between different adrenoceptors in rat hearts and HEK293 cells

There exist α_(1A), α_(1B), α_(1D), β_1, and β_2 subtypes ofadrenoceptors (AR) in rat hearts. Its physiological significancehas not been clear. We investigated the crosstalks between thosecoexisted subtypes in the rat heart and HEK 293 cells. Ourresults indicated: (1) In electric driven isolated rat left atria,either α_(1A~-) or α_(1B~(-AR)) per se induced positive inotropic response(PIR). But when they were artivated together with β-AR, theα_(1A)-AR inihibited, while the α_(1B)-AR potentiated the β-AR mediatedPIR and cAMP accumulation. The α_(1D)-AR did not show aboveeffects. When all the subtypes of α_1-AR were acticated with β-AR, the α_(1A)-AR played a dominated effect. Activation of α_1-ARshowed no effect on the foskolin-induced cAMP accumulation. Italso did not change the binding characteristiics of β-AR. Those     

β-Adrenoceptors potentiate α1-adrenoceptor-mediated inotropic response in rat left atria

AIM: To investigate whether stimulation of β-adrenoceptor (AR) and its subtypes augment α1-AR-evoked positive inotropic response and inositol phosphate (InsP) accumulation in isolated rat left atria. METHODS: Inotropic response was determined by contractile function experiment in isolated electrically driven rat left atria. ^3H-InsP accumulations were measured by ^3H-inositol incorporation and column chromatography. RESULTS: (1) Stimula-tion of α1-AR by phenylephrine (PE) or norepinephrine (NE) in the presence of propranolol (Prop) evoked positive inotropic response and ^3H-InsP accumulations, while stimulation of β-AR by isoprenaline (ISO) or NE in the presence of phentolamine (Phen) only evoked positive inotropic response, but not ^3H-InsP accumulations. (2) Simultaneous stimulation of α1- and β-AR by NE or ISO plus PE significantly shifted the concentration-dependent inotropic response curves and ^3H-InsP accumulation curves to the left and upward compared with individual α1-AR stimulation by PE or NE in the presence of Prop. (3) In the presence of ICI118551 (selective β2-AR antagonist) or CGP12177 (selective β1-AR antagonist), stimulation of either β1- or β2-AR did not change α1-AR-evoked inotropic response and ^3H-InsP accumulations. CONCLUSION: Stimulation of β1-AR and β2-AR potentiates α1-AR-mediated positive inotropic response and InsP accumulation in isolated rat left atria.     

Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell ^3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.     

苯肾上腺素对糖尿病大鼠室性心律失常的诱导作用

目的比较α1肾上腺素受体(α1-AR)激动剂苯肾上腺素对正常及链脲霉素(STZ)诱导的糖尿病大鼠室性心律失常的差异作用,及对两组大鼠左心室肌细胞早期后除极(EAD)的差异作用;并分析两组大鼠左心室α1-AR各亚型的差异表达,解析其可能的物质基础。方法采用离体心脏心电图及电流钳法,分析α1-AR激动剂(苯肾上腺素,PE)诱导正常和糖尿病大鼠离体心脏心律失常发生率及左心室肌细胞EAD发生率的差异作用。采用Westernblot法,比较两组大鼠左心室α1-AR各亚型的蛋白表达。结果与正常大鼠相比,PE(100μmol.L-1)诱导糖尿病大鼠离体心脏产生室性心律失常的机率明显增加,PE(1000μmol.L-1)诱导左心室肌细胞产生EAD的机率明显增加;α1A-AR在糖尿病大鼠左心室的表达明显增加。结论α1-AR激动剂使糖尿病大鼠左心室肌细胞产生高机率的EAD是其诱导高机率室性心律失常的基础。而糖尿病大鼠左心室α1A-AR表达明显增加是其物质基础。     

Dopamine induces intracellular Ca2+ signals mediated by alpha1B-adrenoceptors in rat pineal cells.

We have studied the functional interaction of dopamine with alpha1-adrenoceptor subtypes by measuring intracellular Ca2+ levels in pineal cells, a cell type where adrenoceptors are well characterized. We show that dopamine induces transient intracellular Ca2+ signals in only 70% of cells responding to phenylephrine. Dopamine-induced Ca2+ signals desensitise faster than Ca2+ transients elicited with phenylephrine and are selectively blocked by desipramine, imipramine, and alpha1B-adrenoceptor antagonists. These results suggest that dopamine induced Ca2+ signals are mainly due to the activation of one subtype of alpha-adrenoceptor, the alpha1B.     

Functional α1-adrenergic receptor subtypes in human right gastroepiploic artery

AIM: To study the functional alpha1-adrenergic receptor (alpha1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of alpha2-AR, alpha1-AR, and alpha1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by alpha2-AR selective antagonist yohimbine (pA2 6.82+/-0.28, slope 1.12+/-0.40),alpha1-AR selective antagonist prazosin (pA2 9.77+/-0.22, slope 0.90+/-0.22),alpha1A-AR selective antagonists RS17053 (pA2 8.42+/-0.20, slope 0.93+/-0.20) and 5-MU (pA2 8.42+/-0.22, slope 0.88+/-0.18),alpha1D-AR selective antagonist BMY7378 (pA2 6.84+/-0.32, slope 1.05+/-0.17), and alpha1A-,alpha1B-AR selective antagonist WB4101 (pA2 8.88+/-0.20, slope 1.15+/-0.16). The correlation coefficients between these pA2 values of alpha1-AR selective antagonists with pKi values of which obtained from alpha1A-, alpha1B- and alpha1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an alpha1B- and alpha1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9+/-0.5 to 5.6+/-0.6 and the maximal contraction was changed from (8.9+/-3.2) g to (8.0+/-3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by alpha1-AR, of which alpha1A-AR plays an important role, whereas alpha1B- and alpha1D-AR are not involved in the contraction response.     

Internalization and distribution of three α1-adrenoceptor subtypes in HEK293A cells before and after agonist stimulation

AIM: To examine the subcellular distribution of the 3 alpha1-adrenoceptor (alpha1-AR) subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney 293A cells. METHODS: Confocal real-time imaging, enzyme linked immunosorbent assay (ELISA) and whole cell [3H]-prazosin binding assay were applied to detect the distribution and localization of the 3 alpha1-AR subtypes. RESULTS: alpha1A-AR was found both on the cell surface and in the cytoplasm; alpha1BAR, however, was predominantly detected on the cell surface, while alpha1D-AR was detected mainly in the intracellular compartments. After stimulation with phenylephrine, localization changes were detected by confocal microscopy for alpha1A- and alpha1B-AR,but the localization of alpha1D-AR were unaffected. Phenylephrine stimulation promoted a more rapid internalization of alpha1B-AR than alpha1A-AR. alpha1D-AR internalization was detected only by ELISA. Whole cell [3H]-prazosin binding assay showed that alpha1A-AR functional receptors were detected both on the cell surface and in the cytoplasm; alpha1B-AR, however, were detected predominantly on the cell surface, while alpha1D-AR were detected mainly in intracellular compartments. Phenylephrine stimulation promoted internalization of alpha1A- and alpha1B-AR. CONCLUSION: Phenylephrine stimulation induced changes in the localization of the 3 alpha1-AR.     

Norepinephrine modulates glutamatergic transmission in the bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) and its adrenergic input are key components in stress-induced reinstatement and maintenance of drug use. Intra-BNST injections of either beta-adrenergic receptor (beta-AR) antagonists or alpha2-adrenergic receptor (alpha2-AR) agonists can inhibit footshock-induced reinstatement and maintenance of cocaine- and morphine-seeking. Using electrophysiological recording methods in an in vitro slice preparation from C57/Bl6j adult male mouse BNST, we have examined the effects of adrenergic receptor activation on excitatory synaptic transmission in the lateral dorsal supracommissural BNST (dBNST) and subcommissural BNST (vBNST). Alpha2-AR activation via UK-14,304 (10 microM) results in a decrease in excitatory transmission in both dBNST and vBNST, an effect predominantly dependent upon the alpha2A-AR subtype. Beta-AR activation via isoproterenol (1 microM) results in an increase in excitatory transmission in dBNST, but not in vBNST. Consistent with the work with receptor subtype specific agonists, application of the endogenous ligand norepinephrine (NE, 100 microM) elicits two distinct effects on glutamatergic transmission. In dBNST, NE elicits an increase in transmission (62% of dBNST NE experiments) or a decrease in transmission (38% of dBNST NE experiments). In vBNST, NE elicits a decrease in transmission in 100% of the experiments. In dBNST, the NE-induced increase in synaptic transmission is blocked by beta1/beta2- and beta2-, but not beta1-specific antagonists. In addition, this increase is also reduced by the alpha2-AR antagonist yohimbine and is absent in the alpha2A-AR knockout mouse. In vBNST, the NE-induced decrease in synaptic transmission is markedly reduced in the alpha2A-AR knockout mouse. Further experiments demonstrate that the actions of NE on glutamatergic transmission can be correlated with beta-AR function.     

Alpha1A-adrenergic receptor polymorphism: association with ethnicity but not essential hypertension

The alpha1-adrenergic receptor (alpha1-AR) mediates vasoconstriction and plays an important role in the regulation of vascular tone. Increased alpha1-AR-mediated vasoconstrictor sensitivity, increased vascular reactivity to stress, and an increased prevalence of hypertension occur in African-Americans. The human alpha1A-AR is the predominant alpha1-AR subtype in vascular smooth muscle. The potential relevance of alpha1A-AR genetic variation to ethnic differences in vascular response and to the pathogenesis of hypertension prompted us to determine the frequency distribution of a recently identified polymorphism (Arg492 to Cys) in the alpha1A-AR in normotensive and hypertensive black and white American individuals. Polymerase chain reaction-based PstI restriction fragment length polymorphisms in the human alpha1A-AR gene were determined in 231 African-American and 282 Caucasian individuals, both with and without hypertension. There were marked differences in the genotypic and allelic distributions of the Arg492 to Cys alpha1A-AR polymorphism between African-American and Caucasian individuals (Cys492/Cys492 genotype, normotensive: 7.6% versus 30.1%; hypertensive: 7.1% versus 26.2%; Cys492 allele, normotensive: 29.5% versus 53.8%; hypertensive: 28.8% versus 55.2%; blacks versus whites, P < 0.0001). The frequency of the variant Cys492 allele was similar in normotensive and hypertensive individuals, both in African-Americans (29.5% versus 28.8%) and Caucasians (53.8% versus 55.2%). There were no significant intergenotypic differences in blood pressure (all P > 0.05). The data indicate that this polymorphism is not associated with essential hypertension in black or white Americans, but that the frequency of the alpha1A-AR Arg492 allele occurs significantly more commonly in African-Americans than in Caucasians. The potential role of the Arg492 to Cys alpha1A-AR polymorphism in ethnic differences in vascular alpha1-adrenergic response requires further investigation.     

α1D-Adrenergic receptor insensitivity is associated with alterations in its expression and distribution in cultured vascular myocytes

Aim:

It is unclear why α_1D-adrenergic receptors (α_1D-ARs) play a critical role in the mediation of peripheral vascular resistance and blood pressure in situ but function inefficiently when studied in vitro. The present study examined the causes for these inconsistencies in native α₁-adrenergic functional performance between the vascular smooth muscle and myocytes.

Methods:

The α₁-adrenergic mediated contraction, Ca²⁺ signaling and the subcellular receptor distribution were evaluated using the Fluo-4, BODIPY-FL prazosin and subtype-specific antibodies.

Results:

Rat aortic rings and freshly dissociated myocytes displayed contractile and increased intracellular Ca²⁺ responses to stimulation with phenylephrine (PE, 10 μmol), respectively. However, the PE-induced responses disappeared completely in cultured aortic myocytes, whereas PE-enhanced Ca²⁺ transients were seen in cultured rat cardiac myocytes. Further studies indicated that α_1D-ARs, the major receptor subtype responsible for the α₁-adrenergic regulation of aortic contraction, were distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, similar to the α_1A-AR subcellular localization in the cultured cardiomyocytes. In the cultured aortic myocytes, however, in addition to a marked decrease in their protein expression relative to the aorta, most labeling signals for α_1D-ARs were found in the cytoplasm. Importantly, treating the culture medium with charcoal/dextran caused the reappearance of α_1D-ARs at the cell surface and a partial restoration of the Ca²⁺ signal response to PE in approximately 30% of the cultured cells.

Conclusion:

Reduction in α_1D-AR total protein expression and disappearance from the cell surface contribute to the insensitivity of cultured vascular smooth muscle cells to α₁-adrenergic receptor activation.     

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor

1. The functional coupling of beta(2)-adrenoceptors (beta(2)-ARs) to murine L-type Ca(2+) current (I(Ca(L))) was investigated with two different approaches. The beta(2)-AR signalling cascade was activated either with the beta(2)-AR selective agonist zinterol (myocytes from wild-type mice), or by spontaneously active, unoccupied beta(2)-ARs (myocytes from TG4 mice with 435 fold overexpression of human beta(2)-ARs). Ca(2+) and Ba(2+) currents were recorded in the whole-cell and cell-attached configuration of the patch-clamp technique, respectively. 2. Zinterol (10 microM) significantly increased I(Ca(L)) amplitude of wild-type myocytes by 19+/-5%, and this effect was markedly enhanced after inactivation of Gi-proteins with pertussis-toxin (PTX; 76+/-13% increase). However, the effect of zinterol was entirely mediated by the beta(1)-AR subtype, since it was blocked by the beta(1)-AR selective antagonist CGP 20712A (300 nM). The beta(2)-AR selective antagonist ICI 118,551 (50 nM) did not affect the response of I(Ca(L)) to zinterol. 3. In myocytes with beta(2)-AR overexpression I(Ca(L)) was not stimulated by the activated signalling cascade. On the contrary, I(Ca(L)) was lower in TG4 myocytes and a significant reduction of single-channel activity was identified as a reason for the lower whole-cell I(Ca(L)). The beta(2)-AR inverse agonist ICI 118,551 did not further decrease I(Ca(L)). PTX-treatment increased current amplitude to values found in control myocytes. 4. In conclusion, there is no evidence for beta(2)-AR mediated increases of I(Ca(L)) in wild-type mouse ventricular myocytes. Inactivation of Gi-proteins does not unmask beta(2)-AR responses to zinterol, but augments beta(1)-AR mediated increases of I(Ca(L)). In the mouse model of beta(2)-AR overexpression I(Ca(L)) is reduced due to tonic activation of Gi-proteins.     

Intracellular Ca2+ and adrenergic responsiveness of cardiac myocytes in streptozotocin-induced diabetes

1. The contractile function of diabetic hearts is impaired. In addition, the responsiveness of diabetic cardiac muscle to sympathetic stimulation is altered. Previous studies have revealed a depressed response to beta-adrenoceptor stimulation; however, the response to alpha-adrenoceptor activation remains controversial. Because alpha- and beta-adrenoceptor agonists increase cardiac contractility, largely through increased mobilization of intracellular Ca2+, the aim of the present study was to investigate the effects of alpha- and beta-adrenoceptor stimulation on intracellular Ca2+ handling in cardiac myocytes from streptozotocin-induced diabetic rats. 2. Intracellular Ca2+ was measured using fura-2. Under basal conditions (27 degrees C, 2.5 mmol/L extracellular [Ca2+], 0.3 Hz stimulation), there was no significant difference in resting or peak Ca2+ levels between control and diabetic cardiomyocytes. However, the time course of the intracellular Ca2+ transient was significantly prolonged in cells from diabetic hearts. 3. The beta-adrenoceptor agonist orciprenaline (at 10(-7) and 10(-6) mol/L) increased the amplitude of the Ca2+ transient in both groups; however, the extent of potentiation was less in diabetic compared with control cardiomyocytes. Orciprenaline decreased the duration of the transient to the same extent in both groups. 4. The alpha-adrenoceptor agonist phenylephrine (at 10(-7) and 10(-6) mol/L) had no effect on the Ca2+ transient in control myocytes but caused a significant concentration-dependent increase in its amplitude in diabetic cardiomyocytes. Phenylephrine had no effect on the time course of the transient in either group. 5. These results demonstrate differential effects of insulin-dependent diabetes on the responsiveness of cardiomyocytes to alpha- and beta-adrenoceptor stimulation. The heightened response to alpha-adrenoceptor stimulation observed in diabetic cardiomyocytes may partly compensate for the diminished myocardial beta-adrenoceptor response.