The role of inhibitory heterotrimeric G proteins in the control of in vivo heart rate dynamics

Multiple isoforms of inhibitory Galpha-subunits (Galphai1,2,3, as well as Galphao) are present within the heart, and their role in modulating pacemaker function remains unresolved. Do inhibitory Galpha-subunits selectively modulate parasympathetic heart rate responses? Published findings using a variety of experimental approaches have implicated roles for Galphai2, Galphai3, and Galphao in parasympathetic signal transduction. We have compared in vivo different groups of mice with global genetic deletion of Gialpha1/Galphai3, Galphai2, and Galphao against littermate controls using implanted ECG telemetry. Significant resting tachycardia was observed in Galphai2(-/-) and Galphao(-/-) mice compared with control and Galphai1(-/-)/Galphai3(-/-) mice (P < 0.05). Loss of diurnal heart rate variation was seen exclusively in Galphao(-/-) mice. Using heart rate variability (HRV) analysis, compared with littermate controls (4.02 ms2 +/- 1.17; n = 6, Galphai2(-/-)) mice have a selective attenuation of high-frequency (HF) power (0.73 ms2 +/- 0.31; n = 5, P < 0.05). Galphai1(-/-)/Galphai3(-/-) and Galphao(-/-) cohorts have nonsignificant changes in HF power. Galphao(-/-) mice have a different basal HRV signature. The observed HRV phenotype in Galphai2(-/-) mice was qualitatively similar to atropine (1 mg/kg)-treated controls [and mice treated with the GIRK channel blocker tertiapinQ (0.05 mg/kg)]. Maximal cardioinhibitory response to the M(2)-receptor agonist carbachol (0.5 mg/kg) compared with basal heart rate was attenuated in Galphai2(-/-) mice (0.08 +/- 0.04; n = 6) compared to control (0.27 +/- 0.04; n = 7 P < 0.05). Our data suggest a selective defect of parasympathetic heart rate modulation in mice with Galphai2 deletion. Mice with Galphao deletion also have a defect in short-term heart rate dynamics, but this is qualitatively different to the effects of atropine, tertiapinQ, and Galphai2 deletion. In contrast, Galphai1 and Galphai3 do not appear to be essential for parasympathetic responses in vivo.     

The regulator of G protein signaling RGS4 selectively enhances alpha 2A-adreoreceptor stimulation of the GTPase activity of Go1alpha and Gi2alpha

Agonist-stimulated high affinity GTPase activity of fusion proteins between the alpha(2A)-adrenoreceptor and the alpha subunits of forms of the G proteins G(i1), G(i2), G(i3), and G(o1), modified to render them insensitive to the action of pertussis toxin, was measured following transient expression in COS-7 cells. Addition of a recombinant regulator of G protein signaling protein, RGS4, did not significantly affect basal GTPase activity nor agonist stimulation of the fusion proteins containing Galpha(i1) and Galpha(i3) but markedly enhanced agonist-stimulation of the proteins containing Galpha(i2) and Galpha(o1.) The effect of RGS4 on the alpha(2A)-adrenoreceptor-Galpha(o1) fusion protein was concentration-dependent with EC(50) of 30 +/- 3 nm and the potency of the receptor agonist UK14304 was reduced 3-fold by 100 nm RGS4. Equivalent reconstitution with Asn(88)-Ser RGS4 failed to enhance agonist function on the alpha(2A)-adrenoreceptor-Galpha(o1) or alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins. Enzyme kinetic analysis of the GTPase activity of the alpha(2A)-adrenoreceptor-Galpha(o1) and alpha(2A)-adrenoreceptor-Galpha(i2) fusion proteins demonstrated that RGS4 both substantially increased GTPase V(max) and significantly increased K(m) of the fusion proteins for GTP. The increase in K(m) for GTP was dependent upon RGS4 amount and is consistent with previously proposed mechanisms of RGS function. Agonist-stimulated GTPase turnover number in the presence of 100 nm RGS4 was substantially higher for alpha(2A)-adrenoreceptor-Galpha(o1) than for alpha(2A)-adrenoreceptor-Galpha(i2). These studies demonstrate that although RGS4 has been described as a generic stimulator of the GTPase activity of G(i)-family G proteins, selectivity of this interaction and quantitative variation in its function can be monitored in the presence of receptor activation of the G proteins.     

Heart rate dynamics in iNOS knockout mice

Nitric oxide has both an inhibitory and excitatory role in the regulation of pre-ganglionic sympathetic neurons, involving the iNOS and nNOS systems respectively. The aim of the present study was to examine cardiovascular autonomic activity in iNOS knockout mice using spectral analysis of heart rate variability (HRV), and to determine the role of iNOS in altered HRV in endotoxaemia. Electrocardiograms were recorded in anaesthetised mice, and the R-R intervals digitized for spectral analysis of HRV and cardiac rhythm regularity using sample entropy analysis. The basal heart rate was higher in iNOS knockout mice compared with controls (465+/-8 vs 415+/-13 beat/min P<0.05), with a significant increase in the low frequency power of HRV spectra in iNOS knockout mice compared with controls (49.4+/-4.3 vs 33.8+/-5.6 normalized units, P<0.05), consistent with increased cardiac sympathetic activity. Endotoxaemia is known to decrease HRV, but the role of iNOS is unknown. LPS (20 mg/kg i.p) increased basal heart rate in both wild type and iNOS knockout mice, but caused a depression of HRV and sample entropy in both groups. Studies in isolated beating atria showed that the changes of HRV under basal or post-LPS conditions disappeared in vitro, suggesting that the autonomic system is responsible for altered HRV. We conclude that disruption of iNOS gene leads to an increase in the low frequency power of HRV consistent with increased cardiac sympathetic activity. These data also demonstrate that LPS-induced decrease of HRV is independent of iNOS.     

A new algorithm for wavelet-based heart rate variability analysis

One of the most promising non-invasive markers of the activity of the autonomic nervous system is heart rate variability (HRV). HRV analysis toolkits often provide spectral analysis techniques using the Fourier transform, which assumes that the heart rate series is stationary. To overcome this issue, the Short Time Fourier Transform (STFT) is often used. However, the wavelet transform is thought to be a more suitable tool for analyzing non-stationary signals than the STFT. Given the lack of support for wavelet-based analysis in HRV toolkits, such analysis must be implemented by the researcher. This has made this technique underutilized.This paper presents a new algorithm to perform HRV power spectrum analysis based on the Maximal Overlap Discrete Wavelet Packet Transform (MODWPT). The algorithm calculates the power in any spectral band with a given tolerance for the band's boundaries. The MODWPT decomposition tree is pruned to avoid calculating unnecessary wavelet coefficients, thereby optimizing execution time. The center of energy shift correction is applied to achieve optimum alignment of the wavelet coefficients. This algorithm has been implemented in RHRV, an open-source package for HRV analysis. To the best of our knowledge, RHRV is the first HRV toolkit with support for wavelet-based spectral analysis.     

Blood pressure and heart rate variability response to noninvasive ventilation in patients with exacerbations of chronic obstructive pulmonary disease

Sympathetic activation and parasympathetic withdrawal are commonly observed during acute exacerbations of chronic obstructive pulmonary disease (COPD). We have demonstrated previously that noninvasive positive-pressure ventilation (NPPV) improves parasympathetic neural control of heart rate in patients with obstructive sleep apnea. We hypothesized that NPPV may exert such beneficial effects in COPD as well. Therefore, we assessed the acute effects of NPPV on systemic blood pressure and indexes of heart rate variability (HRV) in 23 patients with acute exacerbations of COPD. The measurements of HRV in the frequency domain were computed by an autoregressive spectral technique. The use of NPPV resulted in significant increases of oxygen saturation (from 89.2+/-1.0 to 92.4+/-0.9 %, p<0.001) in association with reductions in systolic and diastolic blood pressures and heart rate (from 147+/-3 to 138+/-3 mm Hg, from 86+/-2 to 81+/-2 mm Hg, from 85+/-3 to 75+/-2 bpm, p<0.001 for all variables), and increases in ln-transformed high frequency band of HRV (from 6.4+/-0.5 to 7.4+/-0.6 ms(2)/Hz, p<0.01). Reductions in heart rate and increases in ln-transformed HF band persisted after NPPV withdrawal. In conclusion, these findings suggest that NPPV may cause improvements in the neural control of heart rate in patients with acute exacerbations of COPD.     

Increased heart rate variability in mice overexpressing the Cu/Zn superoxide dismutase

Cu/Zn superoxide dismutase (SOD1) is implicated in various pathological conditions including Down's syndrome, neurodegenerative diseases, and afflictions of the autonomic nervous system (ANS). To assess the SOD1 contribution to ANS dysfunction, especially its influence on cardiac regulation, we studied the heart rate variability (HRV) and cardiac arrhythmias in conscious 12-month-old male and female transgenic mice for the human SOD1 gene (TghSOD1). TghSOD1 mice presented heart rate reduction as compared with control FVB/N individuals. All HRV parameters reflecting parasympathetic activity were increased in TghSOD1. Pharmacological studies confirmed that the parasympathetic tone was exacerbated and the sympathetic pathway was functional in TghSOD1 mice. A high frequency of atrioventricular block and premature ventricular contractions was observed in TghSOD1. By biochemical assays we found that SOD1 activities were multiplied by 9 and 4 respectively in the heart and brainstem of transgenic mice. A twofold decrease in cholinesterase activity was observed in the heart but not in the brainstem. We demonstrate that SOD1 overexpression induces an ANS dysfunction by an exacerbated vagal tone that may be related to impaired cardiac activity of the cholinesterases and may explain the high occurrence of arrhythmias.     

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

Heart rate variability in women exposed to very cold air (−110 °C) during whole-body cryotherapy

Heart rate monitoring was used to measure heart rate variability (HRV) at thermoneutral conditions (Ta 24 °C) in healthy women resting in supine position before and after acute and after repeated (3 times a week during a 3-month period) whole-body cryotherapies (WBC), at −110 °C. The observed acute cooling-related increase in high frequency power (HFP) of RR-intervals indicates an increase in cardiac parasympathetic modulation. After 3 months of repeated WBC the increase in parasympathetic tone was attenuated, which may be interpreted as an adaptation of autonomic function. The repeated WBC exposures-related increase in resting low frequency power (LFP) of RR-intervals during the 3 months resembles the response observed related to exercise training.     

The influence of short-term sedentary behavior on circadian rhythm of heart rate and heart rate variability

The notion that sedentary behavior is harmful to human health is widespread. Little is known about the short term influence of sedentary behavior on heart rate (HR) and heart rate variability (HRV) circadian rhythms. Therefore the purpose of the present study was to examine the influence of short term sedentary behavior on the circadian rhythms of HR and HRV using cosine periodic regression analysis. Sixteen healthy young students were included in a randomized crossover study. All subjects underwent 24-h ECG Holter monitoring in two different states of physical activity, an active condition (more than 15,000 steps per day) and a sedentary condition (less than 1,000 steps per day). Hourly mean values were calculated for HR and HRV, and then were evaluated using cosine periodic regression analysis. The circadian rhythm parameters, amplitude, mesor, and acrophase for HR and HRV variables were obtained. As a result, the significance of the circadian rhythm was confirmed for all variables in each condition. The measure of fit R2 value was decreased in sedentary condition. The amplitude of the sedentary condition was significantly smaller than that of the active condition with respect to HR (7.94 ± 1.91 bpm vs. 15.4 ± 3.93 bpm, p < 0.001), natural log of the high frequency measurement (lnHF) (0.38 ± 0.21 ms2 vs. 0.80 ± 0.28 ms2, p < 0.001), and low frequency/high frequency ratio (LF/HF) (0.75 ± 0.54 vs. 1.24 ± 0.69, p = 0.008). We found that sedentary behavior not only significantly lowered the amplitude of HR and HRV variables, but also might have led to weakness of the circadian rhythm of the HR and HRV variables.     

AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin

A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha. The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs. The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha. The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha. The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.     

Comparative analysis of the efficacy of A1 adenosine receptor activation of Gi/o alpha G proteins following coexpression of receptor and G protein and expression of A1 adenosine receptor-Gi/o alpha fusion proteins

HEK293T cells were transiently transfected to express either the human A1 adenosine receptor together with pertussis toxin-resistant cysteine-to-glycine forms of the alpha subunits of Gi1 (C351G), Gi2 (C352G), and Gi3 (C351G) and wild-type Go1alpha or fusion proteins comprising the A1 adenosine receptor and these Gi/o G proteins to compare A1 adenosine receptor agonist-mediated activation of these Gi family G proteins upon coexpression of individual Gi/o G proteins and receptor versus expression as receptor-G protein fusion proteins. Addition of the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) to membranes of pertussis toxin-treated cells resulted in a concentration-dependent stimulation of [35S]GTPgammaS binding with comparable amounts of NECA required to produce half-maximal stimulation following transfection of A1 adenosine receptor and Gi/o G proteins either as fusion proteins or as separate polypeptides. However, the magnitude of agonist-mediated activation of GTPgammaS binding was greatly enhanced by expressing the A1 adenosine receptor and Gi family G proteins from chimaeric open reading frames. This observation was consistent following the study of more than 40 agonists. No preferential activation of any G protein was observed with more than 40 A1 receptor agonists following cotransfection of receptor with G protein or transfection of receptor-G protein fusion proteins. These studies demonstrate the utility of using fusion proteins to study receptor-G protein interaction, show that the A1 adenosine receptor couples equally well to the Gi/o G proteins Gi1alpha, G i2alpha, Gi3alpha, and Go1alpha, and demonstrate that for a range of agonists there is no selectivity for activation of any particular A1 adenosine receptor-Gi/o G protein combination.     

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.     

A pilot investigation of the effect of extremely low frequency pulsed electromagnetic fields on humans' heart rate variability

The question whether pulsed electromagnetic field (PEMF) can affect the heart rhythm is still controversial. This study investigates the effects on the cardiocirculatory system of ELF-PEMFs. It is a follow-up to an investigation made of the possible therapeutic effect ELF-PEMFs, using a commercially available magneto therapeutic unit, had on soft tissue injury repair in humans. Modulation of heart rate (HR) or heart rate variability (HRV) can be detected from changes in periodicity of the R-R interval and/or from changes in the numbers of heart-beat/min (bpm), however, R-R interval analysis gives only a quantitative insight into HRV. A qualitative understanding of HRV can be obtained considering the power spectral density (PSD) of the R-R intervals Fourier transform. In this study PSD is the investigative tool used, more specifically the low frequency (LF) PSD and high frequency (HF) PSD ratio (LF/HF) which is an indicator of sympatho-vagal balance. To obtain the PSD value, variations of the R-R time intervals were evaluated from a continuously recorded ECG. The results show a HR variation in all the subjects when they are exposed to the same ELF-PEMF. This variation can be detected by observing the change in the sympatho-vagal equilibrium, which is an indicator of modulation of heart activity. Variation of the LF/HF PSD ratio mainly occurs at transition times from exposure to nonexposure, or vice versa. Also of interest are the results obtained during the exposure of one subject to a range of different ELF-PEMFs. This pilot study suggests that a full investigation into the effect of ELF-PEMFs on the cardiovascular system is justified.     

The circadian rhythm of heart rate variability

Purpose: In recent years, the measurement of heart rate variability (HRV) has gained ground even outside research settings in everyday clinical and outpatient practice and in health promotion. Methods: Using the search terms “heart rate variability”, “hrv” and “circadian”, a systematic review was carried out in the PubMed database to find original work that analysed the course of HRV parameters over a 24-h period. Results: A total of 26 original studies were found. Almost all the studies detected a circadian rhythm for the HRV parameters analysed. HRV increased during the night in particular and a nighttime peak during the second half of the night was identified. Conclusions: HRV follows a circadian rhythm. But until today, there isn′t any possibility to make quantitative statements about changes over the course of the day for planning short-term measurements. More qualitative studies must be carried in order to close this knowledge gaps.     

心脏疾病中G蛋白的变化

G蛋白是一类重要的信号转导分子,其生理功能是将细胞膜受体所识别的各种细胞外信号同细胞内一系列效应分子偶联起来,引起核基因转录及蛋白质结构和功能的变化。G蛋白在心脏表达的亚型有Gs、Gi/o、Gq／11、G12／13,参与心肌收缩力、心率、心律和心肌细胞生长的调节。本文着重讨论了心脏G蛋白的分类、结构和功能,以及在心肌肥大、心力衰竭、急性心肌缺血和心律失常等心脏疾病中的改变,以加深对这些疾病的发病机制和病理生理过程的认识。     

The effects of specific respiratory rates on heart rate and heart rate variability

In this study respiratory rates of 3, 4, 6, 8, 10, 12, and 14 breaths per minute were employed to investigate the effects of these rates on heart rate variability (HRV). Data were collected 16 times at each respiratory rate on 3 female volunteers, and 12 times on 2 female volunteers. Although mean heart rates did not differ among these respiratory rates, respiratory-induced trough heart rates at 4 and 6 breaths per minute were significantly lower than those at 14 breaths per minute. Slower respiratory rates usually produced higher amplitudes of HRV than did faster respiratory rates. However, the highest amplitudes were at 4 breaths per minute. HRV amplitude decreased at 3 breaths per minute. The results are interpreted as reflecting the possible effects of the slow rate of acetylcholine metabolism and the effect of negative resonance at 3 cycles per minute.     

Phenotypic screening for heart rate variability in the mouse

We developed a technology for heart rate (HR) variability (HRV) analysis in the mouse for characterization of HR dynamics, modulated by vagal and sympathetic activity. The mouse is the principal animal model for studying biological processes. Mouse strains are now available harboring gene mutations providing fundamental insights into molecular mechanisms underlying cardiac electrical diseases. Future progress depends on enhanced understanding of these fundamental mechanisms and the implementation of methods for the functional analysis of mouse cardiovascular physiology. By telemetric techniques, standard time and frequency-domain measures of HRV were computed with and without autonomic blockade, and baroreflex sensitivity testing was performed. HR modulation in the high-frequency component is predominantly mediated by the parasympathetic nervous system, whereas the low-frequency component is under the influence of both the parasympathetic and sympathetic systems. The presented technology and protocol allow for assessment of autonomic regulation of the murine HR. Phenotypic screening for HR regulation in mice will further enhance the value of the mouse as a model of heritable electrophysiological human disease.     

Cortisol release and heart rate variability in horses during road transport

Based on plasma cortisol concentrations it is widely accepted that transport is stressful to horses. So far, cortisol release during transport has not been evaluated in depth by non-invasive techniques such as analysis of salivary cortisol and faecal cortisol metabolites. Transport also causes changes in heart rate and heart rate variability (HRV). In this study, salivary cortisol, faecal cortisol metabolites, heart rate and HRV in horses transported by road for short (one and 3.5 h) and medium duration (8 h) were determined. With the onset of transport, salivary cortisol increased immediately but highest concentrations were measured towards the end of transport (4.1 ± 1.6, 4.5 ± 2.6, 6.5 ± 1.8 ng/ml in horses transported for one, 3.5 and 8 h, respectively). Faecal cortisol metabolite concentrations did not change during transport, but 1 day after transport for 3.5 and 8 h had increased significantly (p < 0.01), reflecting intestinal passage time. Compared to salivary cortisol, changes in faecal cortisol metabolites were less pronounced. Heart rate increased and beat-to-beat (RR) interval decreased (p < 0.05) with the onset of transport. Standard deviation of heart rate increased while root mean square of successive RR differences (RMSSD) decreased in horses transported for 3.5 (from 74 ± 5 to 45 ± 6 ms) and 8 h (from 89.7 ± 7 to 59 ± 7 ms), indicating a reduction in vagal tone. In conclusion, transport of horses over short and medium distances leads to increased cortisol release and changes in heart rate and HRV indicative of stress. The degree of these changes is related to the duration of transport. Salivary cortisol is a sensitive parameter to detect transient changes in cortisol release.     

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.     

RGS protein specificity towards Gq- and Gi/o-mediated ERK 1/2 and Akt activation, in vitro

Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by G(q) and G(i/o) proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate G(q)and G(i/o)-induced ERK and Akt phosphorylation. To isolate G(q)- and G(i/o)-mediated effects, we exclusively expressed muscarinic M(2) or M(3) receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in M(2)-/M(3)- expressing cells through carbachol stimulation. In co-expressions, M(3)/G(q)-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. M(2)/G(i/o) induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on M(2)/G(i/o)-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of G(q)- and G(i/o)-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards G(q) or G(i/o) proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.