DNA synthesis in cultured adult cardiocytes.

Trypsin-dissociated atrial cardiocytes from adult rats were exposed to [3H]thymidine for sequential 24-hour periods from day 2 to day 12 of culture. On day 3 and each day thereafter, cells were prepared for ultrastructural radioautography and examined with an electron microscope. Maximal incorporation occurred on day 5, when 63 percent of the cardiocytes were labeled. Mitotic activity was never present in more than 0.5 percent of the cardiocytes examined. Incorporation of [3H]thymidine and mitosis occurred only in immature cardiocytes characterized by subsarcolemmal primary filaments and Z bands with or without specific granules; more mature cardiocytes were never labeled.

Lymphocytes from spontaneously hypertensive rats exhibit enhanced adenylyl cyclase-Gi protein signaling.

OBJECTIVE: We have previously demonstrated an augmented activation of Gialpha proteins in heart and aorta from spontaneously hypertensive rats (SHRs), which was attributed to an enhanced expression of Gialpha proteins. Since immortalized lymphoblasts derived from lymphocytes of hypertensive patients have been shown to have enhanced Gi activation, the present studies were undertaken to investigate if lymphocytes from SHRs also exhibit enhanced Gi activation and whether this activation is related to enhanced expression of Gi proteins. METHODS: The levels of G-proteins and mRNA were determined by immunoblotting and Northern blotting techniques, using specific antibodies and cDNA probes, respectively. Adenylyl cyclase activity stimulated or inhibited by agonists was determined to examine the functions of G-proteins. RESULTS: The levels of Gialpha-2, Gialpha-3, Gbeta but not of Gs(alpha45) and Gs(alpha47) were significantly increased in lymphocytes from SHRs as compared to their control Wistar Kyoto (WKY) rats. Similarly the mRNA levels of Gialpha-2 and Gialpha-3 were significantly augmented in SHRs as compared to their age-matched WKYs. The increased levels of Gialpha were reflected in increased functions of Gi in SHRs as indicated by increased inhibition of forskolin-stimulated adenylyl cyclase activity by GTPgammaS. The activity of adenylyl cyclase stimulated by GTPgammaS, isoproterenol, NECA, NaF and forskolin was significantly decreased in SHRs as compared to their age-matched WKY rats. On the other hand, inhibitory hormones, atrial natriuretic peptide and angiotensin II inhibited adenylyl cyclase activity to a greater extent in SHRs as compared to their age-matched WKY rats. CONCLUSIONS: These results indicate that lymphocytes from spontaneously hypertensive rats exhibit enhanced Gi activation (function) which may be attributed to the enhanced expression of Gi proteins. It may be suggested that enhanced Gi expression and associated signaling may be one of the factors responsible for enhanced lymphoblasts proliferation observed in hypertension.

Overexpression of Gi-proteins precedes the development of DOCA-salt-induced hypertension: relationship with adenylyl cyclase.

OBJECTIVE: In the present studies, we have investigated if aorta, like heart from deoxycorticosterone acetate (DOCA)-salt hypertensive rats, (HR) also exhibit enhanced expression of G-protein levels and if these alterations occur before or after the development of blood pressure. METHODS: Sprague-Dawley rats treated with DOCA-salt or vehicle for 1, 2, 3 and 4 weeks were used for these studies. The levels of inhibitory guanine nucleotide regulatory proteins (Gi alpha-2, Gi alpha-3) and G beta proteins were determined by immunoblotting, whereas the levels of Gi alpha-2 and Gi alpha-3 and adenylyl cyclase type V enzyme mRNA were determined by Northern-blotting techniques. RESULTS: The blood pressure was significantly increased in DOCA-salt-treated rats as compared to sham-operated rats after 2 to 4 weeks of treatment; whereas no change in blood pressure was observed after 1 week of treatment (prehypertensive state). However, the levels of Gi alpha-2, Gi alpha-3 and G beta proteins and Gi alpha-2 and Gi alpha-3 mRNA were significantly enhanced in hearts and aorta from DOCA-salt treated rats after 1 week of treatment and remained elevated up to 4 weeks of treatment. In addition, the Gi-mediated inhibitions of adenylyl cyclase by Angiotensin II (Ang II) and C-ANF4-23 were also greater in DOCA-salt-treated rats as compared to sham-operated rats after 1 week and longer periods of treatments (2 to 4 weeks). On the other hand, the levels of Gs alpha were not altered up to 2 weeks of DOCA-salt treatment but significantly decreased in rats treated for 3 and 4 weeks. Furthermore, the stimulatory effects of guanine 5'-[gamma-thio]triphosphate (GTP gamma S), isoproterenol and forskolin on adenylyl cyclase were decreased in both hearts and aorta from DOCA-salt-treated rats after 1 to 4 weeks of treatment as compared to sham-operated rats. The mRNA levels of adenylyl cyclase, type V enzyme in hearts from DOCA-salt treated rats were significantly decreased after 3 and 4 weeks of DOCA-salt treatment but not in rats treated for 1 or 2 weeks. CONCLUSIONS: These results indicate that the enhanced expression of Gi alpha-2 and Gi alpha-3 precedes the development of blood pressure in DOCA-salt-induced hypertension. It can thus be suggested that the increased levels of Gi proteins and resulting decreased levels of cAMP may be one of the factors that contribute to the impaired cardiac contractility and increased vascular tone in DOCA-salt hypertension.

Characterization of cardiac myocyte and tissue beta-adrenergic signal transduction in rats with heart failure.

OBJECTIVE: The cellular basis of alterations in beta-adrenergic signal transduction in rats with chronic heart failure (CHF) remains unclear. The aim of the present study was to examine this signal transduction system in isolated ventricular cardiomyocytes of rats with CHF. We focused on changes in the levels of stimulatory (Gs) and inhibitory G-proteins (Gi). METHODS: CHF was induced in male Wistar rats by coronary artery ligation (CAL). Hemodynamic and biochemical parameters were measured 8 weeks after CAL. Alterations in contractile function and Ca(2+) transients via beta-adrenergic receptor signaling of cardiomyocytes isolated from rats with CHF were characterized by simultaneous measurements of cell shortening and fura-2 fluorescence intensity. RESULTS: Coronary artery-ligated rats showed symptoms of CHF, such as decreased contractile function, increased left ventricular volume, decreased chamber stiffness, and about 40% infarct formation of the left ventricle, by 8 weeks after surgery. The contractile function and Ca(2+) dynamics of cardiomyocytes from the rats with CHF remained normal under basal conditions. Only cardiac cell length was increased. The responses of peak shortening, fura-2 fluorescence ratio amplitude, and cAMP content to beta-adrenoceptor stimulation were reduced in cardiomyocytes of the rats with CHF, whereas direct stimulation of adenylate cyclase did not affect the response of these variables. Cardiomyocyte Gsalpha protein was decreased, whereas no changes in Gialpha proteins were seen in these cells. Increases in tissue Gsalpha and Gialpha proteins in the scar zone were detected. The results on tissue levels of collagen and G-proteins in the viable left ventricle appeared to depend on the presence of nonmyocytes. CONCLUSIONS: The results suggest that impaired contractile function of cardiomyocytes is unlikely to account for global LV contractile dysfunction, and that down-regulation of beta-adrenoceptors occurs in cardiomyocytes per se. The difference in changes of G-protein between the cardiomyocyte and myocardial tissue suggests an appreciable contribution of nonmyocytes to myocardial G-protein levels.

Beta-adrenergic signal transduction and contractility in the canine heart after cardiopulmonary bypass.

OBJECTIVE: Impaired beta-adrenergic signal transduction has been proposed as a mechanism contributing to myocardial depression after cardiac surgery. This study determined the changes in the beta-adrenergic system in a model of postoperative myocardial dysfunction induced by myocardial ischaemia and reperfusion under cardiopulmonary bypass (CPB). Those changes were then related to contractility and responsiveness to beta-adrenergic stimulation. METHODS: Four groups of dog hearts were studied: 7 hearts harvested immediately after anaesthesia induction (control group representing the preoperative cardiac condition); 6 hearts harvested after three hours of chest opening by sternotomy (open chest group serving as control for the effects of anaesthesia and surgery); 7 hearts harvested during CPB after 30 minutes of global ischaemia (ischaemia group); and 10 hearts from dogs submitted to one hour of CPB involving 30 minutes of global cardiac ischaemia, harvested 30 minutes after CPB (ischaemia-reperfusion group). Myocardial membranes were prepared to assess: (1) beta-adrenergic receptor density using the radioligand [125I]iodocyanopindolol; (2) GTP-sensitive adenylate cyclase activity and its regulation by isoprenaline and forskolin; (3) G protein levels, using an immunoblotting technique. Ventricular trabeculae or papillary muscles served to assess contractility and responsiveness to isoprenaline. RESULTS: The control and open chest groups had comparable beta-adrenergic receptor density, adenylate cyclase activity and cardiac contractility. In the ischaemia group, the left ventricular membranes had a 55% decrease in receptor density as compared to the controls (P < 0.005), similar GTP-sensitive adenylate cyclase activity and significantly lower adenylate cyclase responses to stimulation with isoprenaline and forskolin. In the ischaemia-reperfusion group, a 144% increase in the left ventricular receptor density was found as compared to the controls (P < 0.005), with a 70% increase in GTP-sensitive adenylate cyclase activity (P < 0.05), a similar adenylate cyclase response to isoprenaline and a 61% increase in response to forskolin (P < 0.005). As compared to the controls, the ischaemia and ischaemia-reperfusion groups had comparable Gs alpha levels, but markedly decreased Gi alpha-2 and Gi alpha-3 levels. The baseline tension of the isolated muscles in the ischaemia and ischaemia-reperfusion groups was comparable, but was 61% and 47% lower than the controls, respectively (P < 0.05). The maximal isoprenaline stimulated tension in the ischaemia and ischaemia-reperfusion groups was 66% and 36% lower than the controls, respectively (P < 0.05 between all groups). CONCLUSIONS: The beta-adrenergic system is severely depressed during global cardiac ischaemia under CPB, but recovers to supranormal values after CPB. However the increased cAMP generation by myocardial membranes after CPB is associated with decreased tension generation by corresponding cardiac muscles. Thus decreased contractility after CPB may be better explained by cellular alterations distal to cAMP generation rather than by changes in the beta-adrenergic system.

Sarcolemmal alterations during the development of genetically determined cardiomyopathy.

The purpose of this study was to identify alterations in specific enzyme and Ca2+ binding activities in cardiac sarcolemmal fractions from UM-X7.1 myopathic Syrian hamsters during the development of cardiomyopathy. Experimental and healthy control animals were examined from 25 to 200 days of age. Sarcolemmal Na+, K+-ATPase activity was depressed in the myopathic hamsters throughout the time course of this study. Sarcolemmal ATP-independent Ca2+ binding was found to be depressed in experimental animals as early as 55 days of age. Ca2+ -stimulated, Mg2+ -dependent ATPase activity was depressed in the experimental animals by 90 days of age and this decrease in enzyme activity was accompanied by a decrease in ATP-dependent Ca2+ binding capacity of the sarcolemmal membranes. Mg2+ -ATPase and Ca2+ -ATPase activities were only affected in the latter stages of the disease (155 to 200 days old). NaF, epinephrine and Gpp(NH)p stimulation of the sarcolemmal adenylate cyclase activity was also observed to be attenuated during the latter stages of the disease. These defects in adenylate cyclase system of the sarcolemmal fraction appeared specific since basal adenylate cyclase activity was not altered at any age studied. The results demonstrate that the earliest lesions in sarcolemmal activity in myopathic hamster heart occur in Na+, K+-ATPase and ATP-independent Ca2+ binding capacity. These defects correspond temporally to the initial stages of cardiac necrotic development in this strain of myopathic hamster.

Fate of [125I]angiotensin II in adrenal zona glomerulosa cells.

Binding and internalization of [125I]angiotensin II (AII) were studied by morphological and biochemical methods in rats in vivo. Light microscope radioautography demonstrated that [125I]AII binds specifically to adrenal zona glomerulosa (ZG) cells. Ultrastructural radioautographic analysis revealed that [125I]AII binds to the cell surface, clusters in coated pits, is internalized in coated vesicles, and is transported by receptosomes to lysosomes in less than 20 min. Biochemical analysis revealed that as much as 40% of the adrenal radioactive uptake behaves as native [125I]AII as shown by electrophoresis, immunoprecipitation and radioligand binding studies. These results indicate that the effects of AII on the secretion of aldosterone by ZG cells are mediated by cell surface phenomena and not by binding to intracellular organelles involved in steroidogenesis. They also indicate that the half-life of AII bound to receptors and internalized seems to be much longer (min) than in the systemic circulation (sec).

Internalization and lysosomal association of [125I]angiotensin II in norepinephrine-containing cells of the rat adrenal medulla.

The morphological localization of [125I]angiotensin II (AII) in the rat adrenal medulla (AM) was studied by light- and electron-microscopic radioautography in vivo. With light microscopy the presence of binding sites for AII in both norepinephrine-containing (NE) and epinephrine-containing (E) cells was confirmed. With electron microscopy, it was found that AII binds to the cell surface of NE cells, is progressively internalized, and is associated with lysosomes and Golgi complex within 20 min, whereas in E cells AII seems to be internalized earlier and recycled back to the cell surface within 5 min without any appreciable association with intracellular organelles. These results suggest different intracellular pathways for AII in NE and E cells of the rat AM.

Aberrant adenylyl cyclase/cAMP signal transduction and G protein levels in platelets from hypertensive patients improve with antihypertensive drug therapy.

We have previously demonstrated a decreased expression of Gi alpha 2 protein in platelets from spontaneously hypertensive rats that was associated with an altered responsiveness of adenylyl cyclase to hormone stimulation and inhibition. In the present studies, we have used platelets from hypertensive patients and examined the hormonal regulation of adenylyl cyclase as well as the levels of G proteins and their modulation by antihypertensive drug therapy. We performed these studies in platelets from four groups of subjects: normotensive subjects (group 1), untreated mildly essential hypertensive patients (group 2), and treated moderately to severely hypertensive patients whose blood pressure was uncontrolled (group 3) or controlled with drug treatment (group 4). GTP gamma S, 5'-(N-ethylcarboxamido)adenosine (NECA), and prostaglandin E1 stimulated adenylyl cyclase activity to a greater extent in hypertensive patients (group 2). This effect was partially corrected (by approximately 50% to 80%) in the patients under antihypertensive drug therapy (groups 3 and 4). In addition, inhibition of adenylyl cyclase mediated by a ring-deleted analogue of atrial natriuretic factor (C-ANF4.23) observed in control normotensive subjects was blunted in hypertensive patients (group 2) and was not corrected in treated patients. Gi alpha levels determined by immunoblotting were in the same range for the four groups, whereas Gi alpha 2 and Gi alpha 3 levels were decreased by 70% and 60%, respectively, in hypertensive patients (group 2) compared with normotensive subjects. Antihypertensive drug therapy (groups 3 and 4) partially restored Gi alpha 2 levels toward normal (group 1) by about 60% and 70%, respectively; however, the reduced Gi alpha 3 levels in group 2 hypertensive patients were not improved in group 3 but were raised toward normal levels in group 4 by about 55%. These results suggest that the altered responsiveness of platelet adenylyl cyclase to hormones in hypertension and the normalization of the response with antihypertensive drug therapy could partly be due to the ability of the latter to modulate Gi alpha protein expression. These effects on platelet function may underlie the beneficial effects of antihypertensive agents on some of the complications of hypertension.

Atrial natriuretic factor in Purkinje fibers of rabbit heart.

The Purkinje fibers of the rabbit false tendons (chordae tendineae spuriae) are endocrine cells containing immunoreactive atrial natriuretic factor (ANF) and ANF messenger RNA (mRNA). These cells, as visualized by immunocryoultramicrotomy, contain immunoreactive ANF in their secretory granules and their Golgi complex and exhibit ANF mRNA, as visualized by in situ hybridization with an ANF complementary RNA probe. The content of immunoreactive ANF and ANF mRNA of the Purkinje fibers is midway between that of atrial and ventricular working cardiocytes. High-pressure liquid chromatography analysis of immunoreactive ANF using antibodies against the C-terminal and N-terminal moieties of the molecule indicates that part of immunoreactive ANF contained in Purkinje fibers is the propeptide [Asn1,Tyr126]ANF whereas part was nonspecifically cleaved into C-terminal and N-terminal ANF. The chordae tendineae spuriae exhibit binding sites for ANF (Kd:approximately 1.0 nM; Bmax:approximately 2.3 fmol/mg). ANF profoundly decreases basal and stimulated (epinephrine, dopamine, isoproterenol, and forskolin) adenylate cyclase activity and cyclic adenosine monophosphate (AMP) levels. ANF has little effect on norepinephrine-stimulated adenylate cyclase activity or on norepinephrine-stimulated cyclic AMP levels. ANF produces only a slight increase in guanylate cyclase activity and cyclic guanosine monophosphate levels at high (10(7)-10(6) M) concentrations. These results suggest an autocrine function for ANF in the modulation of the impulse in the peripheral conduction cells (Purkinje fibers) of the rabbit through changes in second messenger levels.

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.

Inhibitory effect of atrial natriuretic factor on adenylate cyclase activity in adrenal cortical membranes.

The effect of rat synthetic atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in adrenal cortical membranes. Synthetic ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration-dependent manner. The maximum inhibition observed was about 25% with an apparent ki of 5 X 10(-11) to 10(-10) M. Various hormones such as isoproterenol, dopamine, prostaglandin (PGE1) and ACTH-stimulated adenylate cyclase to various degrees and ANF inhibited the stimulatory effect of all these hormones to some degree but never abolished it. In addition, ANF was also able to inhibit the stimulation exerted by forskolin which activates adenylate cyclase by receptor-independent mechanism. This is the first study demonstrating the inhibitory effect of ANF on adrenal cortical adenylate cyclase. From these results it can be suggested that the inhibition of adenylate cyclase may be one of the mechanisms through which ANF exerts the inhibitory effect on steroidogenesis stimulated by various hormones and agents.

C-type natriuretic peptide and brain natriuretic peptide inhibit adenylyl cyclase activity: interaction with ANF-R2/ANP-C receptors.

C-type natriuretic peptide (CNP) and brain natriuretic peptide (BNP) are members of the natriuretic peptide family, which have been shown to interact with ANP-C/ANF-R2 receptors in addition to ANP-B receptor subtypes. The present study was undertaken to investigate if the interaction of CNP and BNP with ANP-C receptors results in the inhibition of adenylyl cyclase activity. CNP and BNP inhibited adenylyl cyclase activity in heart and brain striatal membranes in a concentration dependent manner with an apparent Ki between 0.1 and 1.0 nM. Maximal inhibition observed in heart membranes were about 25% and 35% for BNP and CNP respectively, however the inhibitions in brain striatal membranes were smaller (approximately 20%). The inhibition was dependent on the presence of guanine nucleotides and was attenuated by pertussis toxin treatment. In addition, CNP inhibited the stimulatory effect of isoproterenol on adenylyl cyclase, whereas CNP as well as BNP showed an additive effect with the inhibitory response of angiotensin II on adenylyl cyclase activity. When the combined effect of C-ANF4-23/BNP, C-ANF4-23/CNP and BNP/CNP at optimal concentrations was studied together on adenylyl cyclase activity, the percent inhibition remained the same for C-ANF4-23 and BNP or C-ANF4-23 and CNP, however, an additive inhibitory effect was observed for BNP and CNP. These results suggest that CNP and BNP like C-ANF4-23 interact with ANP-C receptors and result in the inhibition of adenylyl cyclase activity. On the other hand, CNP and BNP interact with the ANP-C receptor, however, the interaction may be different sites or there may be two subpopulations of ANP-C receptors specific for each of the peptides. These results indicate that BNP and CNP, like ANP and C-ANF4-23, inhibit the adenylyl cyclase/cAMP signal transduction system through an inhibitory guanine nucleotide regulatory protein, by interacting with ANP-C receptor subtypes.

Atrial natriuretic peptide-C receptor and membrane signalling in hypertension.

Atrial natriuretic peptide (ANP) regulates a variety of physiological parameters, including the blood pressure and intravascular volume, by interacting with its receptors present on the plasma membrane. ANP receptors are of three subtypes: ANP-A, -B and -C receptors. ANP-A and ANP-B receptors are guanylyl cyclase receptors, whereas ANP-C receptors are coupled to adenylyl cyclase inhibition or phospholipase C activation through inhibitory guanine nucleotide-regulating protein. Unlike other G protein-coupled receptors, ANP-C receptors have a single transmembrane domain and a short cytoplasmic domain of 37 amino acids, the cytoplasmic domain has a structural specificity like those of other single-transmembrane-domain receptors and 37 amino-acid cytoplasmic domain peptide is able to exert is inhibitory effect on adenylyl cyclase. The activation of ANP-C receptor by C-ANP(4-23) (a ring-deleted peptide of ANP) and C-type natriuretic peptide inhibits the mitogen-activated protein kinase activity stimulated by endothelin-3, platelet-derived growth factor and phorbol-12 myristate 13-acetate. C-ANP also inhibits mitogen-induced stimulation of DNA synthesis, indicating that the ANP-C receptor plays a role in cell proliferation through an inhibition of mitogen-activated protein kinase and suggesting that the ANP-C receptor might also be coupled to other signal transduction mechanism(s) or that there might be an interaction of the ANP-C receptor with some other signalling pathways. ANP receptor binding is decreased in most organs in hypertensive subjects and hypertensive animals. This decrease is consistent with there being fewer guanylyl cyclase-coupled receptors in the kidney and vasculature and selective inhibition of the ANP-C receptor in the thymus and spleen. Platelet ANP-C receptors are decreased in number in hypertensive patients and spontaneously hypertensive rats. ANP-A, -B and -C receptors are decreased in number in deoxycorticosterone acetate-salt-treated kidneys and vasculature; however, the responsiveness of adenylyl cyclase to ANP is augmented in the vasculature and heart and is attenuated completely in platelets. These alterations in ANP receptor subtypes may be related to the pathophysiology of hypertension. Several hormones such as angiotensin II, ANP and catecholamines, the levels of which are increased in hypertension, downregulate or upregulate ANP-C receptors and ANP-C receptor-mediated inhibition of adenylyl cyclase. It can be suggested that the antihypertensive action of several types of drugs such as angiotensin converting enzyme inhibitors, angiotensin type 1 receptor antagonists and beta2-adrenergic antagonists may partly be attributed to their ability to modulate the expression and function of the ANP-C receptor.

Enhanced expression of Gi proteins in non-hypertrophic hearts from rats with hypertension-induced by L-NAME treatment.

OBJECTIVE: The objective of the present studies is to investigate if the enhanced expression of Gs alpha protein and their mRNA observed in various models of hypertensive rats is due to the expressed hypertrophy or hypertension. METHODS: Hypertension, in Sprague-Dawley rats was induced by the oral administration of the arginine analog N(omega)-nitro-L-arginine methyl ester (L-NAME) in their drinking tap water for a period of 4 weeks. The control rats were given plain tap water only. The levels of inhibitory guanine nucleotide regulatory proteins (Gi alpha-2, Gi alpha-3), stimulatory guanine nucleotide proteins (Gs alpha) and G beta proteins were determined by immunoblotting, whereas the levels of Gi alpha-2, Gi alpha-3, Gs alpha and adenylyl cyclase type V enzyme mRNA were determined by Northern-blotting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation from [alpha32P]ATP. RESULTS: The systolic blood pressure was enhanced in L-NAME-treated rats compared to control rats (190 +/- 9.2 mmHg versus 121 +/- 6.3 mmHg); however, heart-to-body-weight ratio was not different in two groups. The levels of Gi alpha-2 and Gi alpha-3 proteins and their mRNA were significantly augmented in hearts from L-NAME-treated rats, however, the levels of Gs alpha and G beta were unaltered. In addition, the effect of low concentrations of GTPgammaS on forskolin (FSK)-stimulated adenylyl cyclase activity (receptor-independent functions of Gi alpha) was significantly enhanced in L-NAME-treated rats. However, the inhibitions of adenylyl cyclase exerted by oxotremorine, C-ANP(4-23) and angiotensin II (AII) (receptor-dependent function of Gi alpha) were completely attenuated in L-NAME-treated rats. On the other hand, cholera toxin stimulated GTP or GTPgammaS-sensitive adenylyl cyclase activity (Gs alpha function) to similar extent in control and L-NAME-treated rats, suggesting that Gs alpha functions were not altered by L-NAME treatment. However, the stimulatory effects of isoproterenol, glucagon, NaF on adenylyl cyclase were diminished in L-NAME-treated rats. In addition, FSK-stimulated enzyme activity was also diminished in L-NAME-treated rats without any changes in the mRNA levels of type V enzyme. CONCLUSIONS: These results suggest that L-NAME hypertensive rats that do not express cardiac hypertrophy exhibit enhanced expression of Gi alpha protein and associated adenylyl cyclase activity.

Altered expression of Gi-protein and adenylyl cyclase activity in hearts from one kidney one clip hypertensive rats: effect of captopril.

OBJECTIVE: To investigate whether one kidney one clip (1K-1C) hypertensive rats associated with high levels of angiotensin II (Ang II) exhibit enhanced expression and functions of G proteins in the heart and whether the enhanced expression can be attributed to Ang II. METHODS: The levels of G protein and G protein mRNA in hearts from 1K-1C hypertensive rats were determined by immunoblotting and Northern blotting techniques using specific antibodies and cDNA probes, respectively, for different isoforms of G proteins. Adenylyl cyclase activity, stimulated or inhibited by agonists, was determined to examine the function of G proteins. RESULTS: The levels of Gialpha-2 and Gialpha-3 proteins and mRNA were significantly increased in hearts from 1K-1C hypertensive rats compared with control rats, whereas the levels of Gsalpha were unchanged. Guanosine 5'-[3'-thio] triphosphate (GTPgammaS), isoproterenol, glucagon, sodium fluoride (NaF) and forskolin (FSK) stimulated adenylyl cyclase activity in hearts from control and hypertensive rats to varying degrees; however, the stimulations were significantly less in hypertensive rats compared with control rats. On the other hand, the inhibitory effect of low concentrations of GTPgammaS on FSK-stimulated adenylyl cyclase activity (an index of Gi function) was significantly enhanced in hearts from 1K-1C hypertensive rats, whereas the inhibitory effect of C-ANF4-23 on adenylyl cyclase was increased and that of Ang II was decreased in hearts from 1K-1C hypertensive rats. Captopril, an angiotensin-converting enzyme inhibitor, restored the augmented levels of Gi proteins and also the altered stimulation and inhibition of adenylyl cyclase by GTPgammaS, stimulatory and inhibitory hormones, respectively, in hearts from hypertensive rats. CONCLUSION: These data suggest that 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions that may be attributable to the enhanced levels of Ang II in this model of hypertension.

Atrial natriuretic factor in the vena cava and sinus node.

We investigated the localization of atrial natriuretic factor (ANF) mRNA and of immunoreactive ANF in the vena cava and sinus node of rat and, for comparative purposes, in atria and ventricles. In situ hybridization with an ANF cRNA probe revealed that the supradiaphragmatic portion of the inferior vena cava contains almost as much mRNA as the atria, whereas the levels were less in the superior vena cava and higher than in ventricles in the sinus node. Immunoreactive ANF (high Mr form) was found to be 22 times less abundant in the supradiaphragmatic vena cava and 148 times less abundant in the superior vena cava than in atrial cardiocytes. The wall of the supradiaphragmatic portion of the vena cava and the valve (eustachian valve) that separates the atrial cavity from that of the vein are made up of atrial-like cardiocytes containing secretory granules. The subendothelial area of the superior vena cava also contains atrial-like cardiocytes with secretory granules, whereas the outer portion of the vein is made up of "transitional cells" without or with only a few secretory granules. Secretory granules in the vena cava and nodal cells, as well as transitional cells, contain immunoreactive ANF. With immunocryoultramicrotomy, virtually all cells, whether atrial-like, transitional, or nodal, and even those without secretory granules, were found to contain immunoreactive ANF in their Golgi complex and in secretory vesicles in the vena cava and in the sinus node.

Angiotensin II receptors negatively coupled to adenylate cyclase in rat myocardial sarcolemma. Involvement of inhibitory guanine nucleotide regulatory protein.

The effect of angiotensin II (AII) on adenylate cyclase was studied in the rat and rabbit heart sarcolemma. AII inhibited adenylate cyclase activity in the rat and rabbit sarcolemma in a concentration-dependent manner. Maximal inhibition of about 35-40% was observed in the rat, with an apparent Ki of about 3 nM; about 30% inhibition, with an apparent Ki of about 6 nM, was noted in rabbit sarcolemma. The inhibitory effect of AII was dependent on the presence of guanine nucleotides and was blocked by saralasin. In addition, AII also inhibited the stimulatory effects of isoproterenol and glucagon on adenylate cyclase. Ninhibin, a sperm factor which has been shown to modify the characteristics of inhibitory guanine nucleotide regulatory protein (Gi), attenuated the inhibitory effects of AII on basal and hormone-sensitive adenylate cyclase. Furthermore, pertussis toxin (PT) treatment of the sarcolemma in the presence of [32P]NAD resulted in ADP-ribosylation of a single 41-kD protein. PT also attenuated the AII-mediated inhibition of basal and hormone-sensitive adenylate cyclase and enhanced the magnitude of the stimulatory effects of isoproterenol and glucagon on adenylate cyclase activity. These data suggest that the rat myocardial sarcolemma contains AII receptors that are negatively coupled to adenylate cyclase through Gi protein.

Altered responsiveness of adenylate cyclase to adenosine and other agents in the myocardial sarcolemma and aorta of spontaneously-hypertensive rats.

Adenylate cyclase activity was studied in the myocardial sarcolemma and aorta of spontaneously-hypertensive rats (SHR) and their respectively Wistar-Kyoto (WKY) controls. Basal enzyme activity was decreased in the SHR as compared to the WKY group. Adenylate cyclase stimulation by N-ethylcarboxamide adenosine (NECA) was significantly lower in the myocardial sarcolemma and aorta of SHR, and this decreased responsiveness was associated with a reduction in the Vmax. Other agonists, such as isoproterenol (ISO), epinephrine, dopamine (DA), and glucagon, also enhanced myocardial adenylate cyclase activity to various degrees in SHR and WKY, but stimulation (Vagonists/Vbasal) was always lower in the SHR. NaF and forskolin (FSK), which activate adenylate cyclase via receptor-independent mechanisms, augmented it in the myocardial sarcolemma of SHR to a lesser extent than in WKY. While the guanine nucleotides GTP and GMP-P(NH)P elevated adenylate cyclase in a concentration-dependent manner in both SHR and WKY, the magnitude of stimulation was significantly lower in the former group. Decreased basal adenylate cyclase activity and responsiveness to adenosine, various hormones, NaF and FSK were observed in SHR of all ages, i.e. from 4 to 24 weeks of age. In addition, basal, hormone-, NaF- and FSK-stimulated adenylate cyclase activity was diminished markedly in the aorta of SHR. These results suggest that, in SHR, not only is basal adenylate cyclase activity decreased but the abilities of adenosine, other hormones and agonists, such as NaF and FSK, to stimulate adenylate cyclase, guanine nucleotide regulatory protein and the catalytic subunit of the cyclase system are also impaired in the myocardial sarcolemma and aorta.

Streptozotocin-induced diabetes and hormone sensitivity of adenylate cyclase in rat myocardial sarcolemma, aorta and liver.

Adenylate cyclase activity was investigated in myocardial sarcolemma, aorta particulate fractions, and liver plasma membranes from control and 5-day streptozotocin-induced diabetic rats. The basal adenylate cyclase activity was increased in heart sarcolemma from diabetic rats, whereas the extent of stimulation by glucagon, dopamine, isoproterenol, epinephrine, sodium fluoride and forskolin was decreased markedly. The decreased responsiveness was associated with a decrease in Vmax but not in the activation constant. In contrast, GTP stimulated adenylate cyclase in control and diabetic myocardial sarcolemma to the same extent. In addition, the basal adenylate cyclase activity was not altered significantly in aorta particulate fraction of liver plasma membranes from diabetic rats, but the stimulation of adenylate cyclase by catecholamines and forskolin (in the case of aorta) and by adenosine, glucagon, NaF and forskolin (in the case of liver) was diminished markedly. These data suggest that, in streptozotocin-induced diabetes, the responsiveness of adenylate cyclase to various hormones and agents (fluoride and forskolin) which act through receptor-independent mechanisms is decreased.