Volume overload cardiac hypertrophy exhibits decreased expression of g(s)alpha and not of g(i)alpha in heart

We haverecently reported enhanced levels of G_i proteins ingenetic and other experimentally induced models of hypertension, whereas the levels of G_s were decreased in hypertensiverats expressing cardiac hypertrophy. The present studies wereundertaken to investigate whether the decreased levels ofG_s are associated with cardiac hypertrophy per se andused an aortocaval fistula (AV shunt; volume overload) rat model thatexclusively expresses cardiac hypertrophy. Cardiac hypertrophy inSprague-Dawley rats (200-250 g) was induced under anesthesia, and,after a period of 10 days, the hearts were used for adenylyl cyclaseactivity determination, protein quantification, and mRNA leveldetermination. A temporal relationship between the expression ofG_s proteins and cardiac hypertrophy was also examined ondays 2, 3, 7, and 10 after induction of AV shuntin the rat. The heart-to-body-weight ratio (mg/g) was significantlyincreased in AV shunt rats after 3, 7, and 10 days of induction of AVshunt compared with sham-operated controls, whereas arterial bloodpressure was not different between the two groups. Guanosine5'-O-(3-thiotriphosphate) (GTPS) stimulated adenylylcyclase activity in a concentration-dependent manner in heart membranesfrom both groups; however, the degree of stimulation was significantlydecreased in AV shunt rats. In addition, the stimulatory effects ofisoproterenol were also diminished in AV shunt rats compared withcontrol rats, whereas glucagon-stimulated adenylyl cyclase activity wasnot different in the two groups. The inhibitory effects of oxotremorine(receptor-dependent G_i functions) and low concentrations ofGTPS on forskolin-stimulated adenylyl cyclase activity(receptor-independent G_i functions) were not different inthe two groups. In addition forskolin and NaF also stimulated adenylylcyclase activity to a lesser degree in AV shunt rats compared withcontrol rats. The levels of G_i-2 and G_i-3proteins and mRNA, as determined by immunoblotting and Northernblotting, respectively, were not different in both groups; however, thelevels of G_s45 andG_s47, and not ofG_s52, proteins were significantly decreasedin AV shunt rats by days 7 and 10 compared withcontrol rats, whereas no change was observed on days 2 and3 after induction of AV shunt. These results suggest thatthe decreased expression of G_s proteins may not be thecause but the effect of hypertrophy and that the diminishedresponsiveness of adenylyl cyclase to GTPS, isoproterenol, NaF, andforskolin in hearts from AV shunt rats may partly be due to thedecreased expression of G_s. It can be concluded fromthese studies that the decreased expression of G_s may beassociated with cardiac hypertrophy and not with arterial hypertension.

     

Hydrogen peroxide enhances the expression of Giα proteins in aortic vascular smooth cells: role of growth factor receptor transactivation

Oxidative stress has been shown to increase the expression of G(i)α proteins in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats. The present study was undertaken to examine if H(2)O(2), which induces oxidative stress, could also enhance the expression of G(i)α proteins in VSMC and to further explore the underlying signaling pathways responsible for this response. Treatment of VSMC with H(2)O(2) increased the expression of G(i)α proteins and not of G(s)α protein in a concentration- and time-dependent manner. A maximal increase of ～40-50% was observed at 100 μM and 1 h and was restored to control levels by AG1295 and AG1478, inhibitors of epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R), respectively, and PD98059 and U126, inhibitors of extracellular signal-regulated kinase (ERK1/2), and wortmannin and AKT inhibitor VIII, inhibitors of PKB/AKT, respectively. In addition, H(2)O(2) also increased the phosphorylation of EGF-R, PDGF-R, ERK1/2, and AKT, which was attenuated by the respective inhibitors, whereas the inhibitors of EGF-R and PDGE-R also inhibited the enhanced phosphorylation of ERK1/2 and AKT. Furthermore, transfection of cells with short interfering RNA of EGF-R and PDGF-R restored the H(2)O(2)-induced enhanced expression of G(i)α proteins to control levels. The increased expression of G(i)α proteins was reflected in enhanced G(i) functions as demonstrated by enhanced inhibition of adenylyl cyclase by inhibitory hormones and forskolin-stimulated adenylyl cyclase activity by a low concentration of GTPγS, whereas G(s)α-mediated stimulations of AC were significantly decreased. Furthermore, H(2)O(2)-induced enhanced proliferation of VSMC was attenuated by dibutyryl-cAMP. These results suggest that H(2)O(2) increases the expression of G(i)α proteins in VSMC through the transactivation of EGF-R/PDGF-R and ERK1/2 and phosphatidylinositol-3 kinase signaling pathways.     

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.     

Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes

We have studied the effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity in cultured cardiocytes from atria (left and right) and ventricles from neonatal rats. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration dependent manner in cultured atrial (right and left atria) and ventricular cells. However the inhibition was greater in atrial cells as compared to ventricular cells. The maximal inhibition observed in ventricular cells was about 35% with an apparent Ki of about 10(-10) M, whereas about 55% inhibition with an apparent Ki between 5 X 10(-10) M and 65% inhibition with an apparent Ki of 10(-9) M were observed in right and left atrial cardiocytes respectively. The inhibitory effect of ANF was dependent on the presence of guanine nucleotides. Various hormones and agents such as isoproterenol, prostaglandins, adenosine, forskolin and sodium fluoride stimulated adenylate cyclase activities to various degrees in these atrial and ventricular cardiocytes. ANF inhibited the stimulatory responses of all these agonists, however the degree of inhibition varied for each agent. In addition ANF also inhibited cAMP levels in these cells. These data indicate that ANF receptors are present in cardiocytes and are negatively coupled to adenylate cyclase.     

Altered expression of G-protein mRNA in spontaneously hypertensive rats.

We have recently demonstrated that the decreased ability of hormones, forskolin and GTP to stimulate adenylate cyclase in heart and aorta from spontaneously hypertensive rats (SHR), as compared to their age-matched Wistar-Kyoto control rats (WKY), was associated with enhanced levels of Gi- and not with Gs-regulatory proteins. In the present studies we have investigated the expression of Gi-regulatory proteins at the mRNA level by Northern blotting. Total RNA of heart ventricle and aorta from WKY and SHR was probed with radiolabeled cDNA inserts encoding Gi alpha-2 and Gi alpha-3. The Gi alpha-2 and Gi alpha-3 probes detected a message of 2-3 and 3-5 kb, respectively, in both WKY and SHR, however, the message was significantly enhanced in SHR, as compared by WKY. On the other hand the cDNA probe encoding Gs alpha detected a message of 1.8 kb in heart and aorta from both WKY and SHR, however, no difference in the levels of Gs alpha mRNA was detected in SHR and WKY tissues. These results indicate that the mRNA levels of Gi alpha-2 and Gi alpha-3 and not of Gs are overexpressed in heart and aorta from SHR, which may be responsible for the increased levels of Gi as shown earlier by immunoblotting techniques. It may be suggested that the enhanced vascular tone and impaired cardiac contractility in hypertension may partly be the consequences of increased levels of Gi in heart and aorta.     

Progress and problems in understanding the involvement of calcium in heart function

In this article we have briefly reviewed the role of Ca2+ in the excitation contraction coupling in the myocardium and have indicated that cardiac contraction and relaxation are initiated upon raising and lowering the intracellular concentration of free Ca2+, respectively. Different mechanisms for the entry of Ca2+ through sarcolemma as well as release of Ca2+ from sarcoplasmic reticulum and possibly mitochondria have been outlined for initiating cardiac contraction. Relaxation of the cardiac muscle appears to be intimately dependent upon efflux of Ca2+ through sarcolemma as well as sequestration of Ca2+ by the intracellular storage sites, particularly sarcoplasmic reticulum and possibly mitochondria. The actions of some pharmacological and pathophysiological interventions have been explained on the basis of changes in subcellular Ca2+ movements in myocardium. Quinidine, which produced an initial positive inotropic action on rat heart was also found to increase sarcolemmal Ca2+-ATPase activity without any changes in the Na+-K+ ATPase. Other antiarrhythmic agents, procainamide and lidocaine, also increased sarcolemmal Ca2+-ATPase activity without affecting the Na+-K+ ATPase. On the other hand, both Ca2+-ATPase and Na+-K+ ATPase activities were increased in heart sarcolemma obtained from cardiomyopathic hamsters. In this model the increased Ca2+-ATPase activity may promote the occurrence of intracellular Ca2+ overload in the cardiac cell whereas the increased Na+-K+ ATPase activity may increase Ca2+ efflux through Na+-Ca2+ exchange systems as an adaptive mechanism. It has been suggested that some caution should be exercised while interpreting the data from in vitro experiments in terms of functional changes in the myocardium. Furthermore, it has been proposed that the pathophysiology and pharmacology of Ca2+ movements at different membrane sites be understood fully in normal and diseased myocardium in order to improve the therapy of heart disease.     

Proffered papers 9.30–10.00 Monday 15 September 2003

Both the original Bethesda system and the current UK classifications of cervical cytology have proved robust but each has a major weakness in the area of abnormalities of uncertain significance. Cytologists recognize that sometimes it is simply impossible to differentiate between reactive and dyskaryotic material. For this reason, the Australian version of the Bethesda system introduced a new category of 'high grade inconclusive' with a recommendation for referral to colposcopy. Approximately 60% of such cases are found to have high grade lesions at colposcopy (Schoolland M, Sterrett G, Knowles S et al .). The present UK system even with the proposed changes requires of the pathologist, a decision as to whether such cases are probably high grade (=a report of moderate dyskaryosis) or not (= a report of borderline). This continues to ignore the fact that sometimes you just cannot tell, even on review. We have taken a consecutive series of 50 referral smears, reported as moderate dyskaryosis, where the histological outcome (by loop cone) is known. These cases were rescreened and then reviewed blind by a pathologist with extensive experience of the Australian NH & MRC modified Bethesda system. On review, the material was reclassified along NH & MRC lines. The results were compared with the biopsy findings in order to determine whether the category of 'inconclusive' might be of value in the context of the NHSCSP.