Stimulation-enhanced 3-O-methylglucose efflux from the frog sartorius: kinetics and properties of the system.

The characteristics of the process by which contraction enhances glucose transport in the frog sartorius were studied. Electrical stimulation increased the permeability of muscles to 3-O-methylglucose (3-O-MeGlc), a nonmetabolizable glucose analogue, increasing efflux as well as uptake. Enhanced efflux was due to an increase in Vmax of the efflux process. A lactacidosis had no effect on basal 3-O-MeGlc efflux, and replacement of media Na+ with Li+ did not affect stimulation-induced uptake. Also, basal and stimulated uptake was not affected by 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator. Lastly, N-carbobenzoxy-glycyl-L-phenylalaninamide, which inhibits insulin-enhanced, but not basal, glucose uptake in adipocytes, inhibited both basal and stimulated 3-O-MeGlc fluxes in the frog sartorius. From these findings, we conclude: (1) contraction and exercise enhance glucose transport in muscle by increasing the number of transporters in the plasma membrane, or their turnover, by an unknown process; and (2) basal glucose transport of muscle, unlike that of adipocytes, can not be distinguished from stimulated transport on the basis of its insensitivity to N-carbobenzoxyglycyl-L-phenylalaninamide.

pCMBS-induced swelling of dogfish (Squalus acanthias) rectal gland cells: role of the Na+,K(+)-ATPase and the cytoskeleton.

(1) 0.1-1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark (Squalus acanthias) rectal glands, with an uptake of cell Na+ and a loss of K+. In contrast, 1 mM N-ethylmaleimide (NEM) does not swell rectal gland cells (RGC), while affecting cell cations. (2) The slow entry of [203Hg]pCMBS is linearly related to its external concentration (10 microM-1 mM) and a small accumulation of pCMBS (apparent gradient about 3) in the cells occurs in 2 h. Cell 203Hg rapidly washes out of the cells (fast rate constant 0.153.min-1; slow rate constant 0.0067.min-1), and this efflux is accelerated by 1mM dithiothreitol. Thus, a major portion of pCMBS inter-acts rather loosely with cell components. (3) pCMBS and NEM share: (a) a negligible effect on the efflux of 86Rb+ and of [14C]urea; (b) a gradual inhibition of the cell Na+,K(+)-ATPase activity. (4) NEM as well as agents lowering cell glutathione accelerate and increase the pCMBS-induced cell swelling. Conditions inhibiting the Na+,K(+)-ATPase (ouabain, absence of Na+) have the same effect. (5) pCMBS, but not NEM produce a disappearance of the F-actin-phalloidin fluorescence independent of cell volume changes, particularly at the basolateral RGC membrane. (6) The data are consistent with the following set of events: (a) pCMBS (but not NEM) affects the cell membrane by increasing the efflux of the cell osmolyte taurine (Ziyadeh et al. (1988) Biochim. Biophys. Acta 943, 43-52 and unpublished data); (b) on entry into the cells, pCMBS and NEM interact with cell -SH, including those of the Na+,K(+)-ATPase; this action produces the observed changes in cell cations. Also, pCMBS, but not NEM, decrease F-actin at the membrane; (c) the inhibition of the Na+,K(+)-ATPase activity together with the decreased resistance of the cell membrane to stretch (absence of F-actin) produces the observed pCMBS-induced cell swelling by osmotic forces (intracellular non-diffusible anions).

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics.

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.

Molecular signalling mechanisms controlling growth and function of cardiac fibroblasts.

Cardiac fibroblasts appear to be important in producing and maintaining the extracellular matrix (ECM) of the heart. The abnormal proliferation of cardiac fibroblasts and deposition of the ECM protein, collagen, associated with hypertension and myocardial infarction, may adversely affect the performance of the heart. Several groups of factors affect collagen gene expression and/or growth of cardiac fibroblasts. Angiotensin II, aldosterone and endothelins play a central role in the remodeling of the ECM in hypertension, and decrease collagenase activity and/or increase collagen synthesis in cultured cells. Regulatory peptides that are generally elevated at sites of injury, such as TGF-beta 1 and PDGF, increase collagen synthesis and/or stimulate mitogenesis. Mechanical stretch enhances collagen expression and cell proliferation, responses which could in part be due to integrin activation. Cytokines may stimulate or inhibit cell growth, the latter through prostaglandin formation. Angiotensin II is a principal determinant in vivo of cardiac fibroplasia and synthesis of the ECM proteins, collagen and fibronectin. Cardiac fibroblasts possess G-protein-coupled AT1 receptors for angiotensin II that couple to activation of multiple signalling pathways, including: phospholipase C-beta, with the subsequent release of Ca2+ from intracellular stores and activation of protein kinase C, mitogen-activated protein kinases, tyrosine kinases, phospholipase D, phosphatidic acid formation, and the STAT family of transcription factors. Cardiac fibroblasts respond to angiotensin II with hyperplastic/hypertrophic growth, and increased expression of collagen, fibronectin, and integrins. The mechanisms by which the AT1 receptor activates multiple signalling pathways are not known, although the receptor might interact at some level with both integrins and cytokine receptors. Different signalling pathways of the AT1 receptor may subserve different cellular responses, such as mitogenesis, ECM synthesis, or an inflammatory/stress response. Crosstalk among the signalling pathways of the AT1 receptor, and those of G-protein, cytokine, and growth-factor receptors, may determine the ultimate response of the cell.

Angiotensin-II-binding sites on hepatocyte nuclei.

Angiotensin-II (Ang II) stimulates gene expression and cell growth in several cell types. Studies that have shown localization of Ang II to nuclei of myocytes and hepatic nuclear Ang II binding suggest that these actions may be mediated by nuclear receptors. We characterized Ang II binding to rat liver nuclei, which were free of plasma membrane based on enzyme analysis and electron microscopy. At 18 C, specific binding of 0.1-0.3 nM [125I]Ang II to nuclei and nuclear envelopes reached equilibrium by 2 h. Unlabeled Ang II inhibited [125I]Ang II binding to nuclei with an IC50 of 1.4 +/- 0.2 nM (+/- SE; n = 6). In half of the nuclear preparations, a lower affinity site (IC50, 50.4 +/- 23.6 nM), which accounted for 7-32% of specific Ang II binding, was detected by Scatchard analysis. Results similar to these were obtained with nuclear envelopes. Other Ang peptides competed for binding in the rank order: Ang III (IC50, 2.1 nM) greater than Ang I (IC50, 33) greater than [Des-Phe8]Ang II (IC50, 362) greater than [Des-Asp1-Des-Arg2]Ang II (IC50, 736). Losartan (DuP 753), an AT1 receptor antagonist, inhibited binding (IC50, 10.9 +/- 0.9 nM), whereas the AT2 receptor antagonist PD123177 did not. The pH optimum for binding to nuclear envelopes was 7, with binding more sensitive to low (5 and 6) than high (8 and 9) pH. Nonhydrolyzable GTP analogs accelerated displacement of bound [125I]Ang II by 10(-5) M Ang II. Differences were noted in pH sensitivity, time course, binding affinity for Ang I, II, and III, and rate of dissociation between nuclei or nuclear envelopes and plasma membrane Ang II binding. These results suggest that nuclear envelopes have a G-protein-coupled Ang II-binding site, which belongs to the AT1 class of Ang II receptors, with properties different from the plasma membrane receptor.

Role of type 1 and type 2 angiotensin receptors in angiotensin II-induced cardiomyocyte hypertrophy.

We compared the ability of angiotensin II (Ang II) to induce hypertrophy of neonatal rat ventricular myocytes with that of endothelin-1. Over 72 hours, Ang II (1 mumol/L) increased the ratio of protein to DNA by less than 10%, whereas endothelin-1 (100 nmol/L) produced a 28% increase. The growth effects of either agonist occurred independently of chronotropic actions. Radioligand binding studies showed that myocytes have nearly 300-fold more receptors for endothelin-1 than Ang II, and type 1 and type 2 Ang II receptor subtypes (AT1 and AT2) are present in near equal proportions. Cotreatment with a 10-fold molar excess of AT2 antagonists (PD 123177 or CGP 42112) for 72 hours augmented the Ang II-induced increase in the protein-to-DNA ratio to levels nearly as high (23%) as those with endothelin-1 (28%). AT2 antagonists enhanced Ang II stimulation of protein synthesis, as indexed by [3H]leucine incorporation, whereas an AT1 antagonist blocked Ang II-induced incorporation. An AT2 antagonist also prevented Ang II-induced protein degradation. In conclusion, Ang II-induced myocyte growth is tempered because of low AT1 levels and an antigrowth effect of AT2. These findings have potential clinical significance in that regression of hypertension-induced cardiac hypertrophy by AT1 antagonists may be in part due to an unopposed antigrowth effect of Ang II mediated via AT2.

Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3 : an autocrine loop for hypertrophy.

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin-angiotensin system.

Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin- released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways.

Protein kinase C in angiotensin II signalling in neonatal rat cardiac fibroblasts. Role in the mitogenic response.

The mitogenic effects of angiotensin II on cardiac fibroblasts are mediated by membrane receptors that are classified as AT1. These receptors are prototypical of the seven transmembrane group of receptors that couple, via G-proteins, to phospholipase C, thereby generating the endogenous activator of protein kinase C, diacylglycerol. Phorbol ester activators of protein kinase C exhibit growth-promoting effects in many cell types, suggesting that this enzyme may be responsible for the growth effects of angiotensin II on cardiac fibroblasts. Both kinase assays and Western analysis demonstrated that angiotensin II does induce translocation of protein kinase C to the detergent-soluble, membrane compartment of cardiac fibroblasts. Although translocation is commonly interpreted to mean activation of protein kinase C, in situ assays on permeabilized cells failed to detect increased enzymatic activity in response to angiotensin II. Nonetheless, this hormone did activate protein kinase C, leading to activation of mitogen-activated protein (MAP) kinases. However, a PKC-independent pathway for activation of MAP kinases exists as well. Downregulation and inhibitor studies indicated that protein kinase C is not critically involved in angiotensin II-induced thymidine incorporation into DNA. Furthermore, phorbol esters that activate protein kinase C do not elicit a mitogenic response in these cells. In conclusion, the mitogenic effects of angiotensin II on cardiac fibroblasts are not simply explained by activation of protein kinase C.

Amplification of angiotensin II signaling in cardiac myocytes by adenovirus-mediated overexpression of the AT1 receptor.

Low levels of AT1 receptor can make studying the growth-related signal transduction events mediated by this angiotensin II receptor in cardiac myocytes technically difficult. The purpose of the present study was to establish whether an adenovirus expression system could be used to increase the number of plasma membrane AT1 receptors in neonatal rat ventricular myocytes, thereby amplifying the signaling pathways activated by this receptor. Cardiac myocytes infected with adenovirus expressing the AT1 receptor exhibited increased ligand binding. The overexpressed receptor appeared to function like the endogenous receptor, in regard to agonist-induced internalization, as well as coupling to MAPK activation and protein tyrosine phosphorylation events. In addition, adenovirus-mediated overexpression of the AT1 receptor resulted in the amplification of angiotensin II intracellular signaling. In conclusion, adenovirus-mediated overexpression of angiotensin II receptors appears to be a useful strategy for studying the signal transduction events activated by this hormone in cardiac myocytes and for unraveling the molecular means by which this receptor type couples to a hypertrophic pattern of growth and gene expression.

Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor.

Three putative protein kinase C phosphorylation sites in the carboxyl-terminal region of the angiotensin II AT1A receptor suggest that protein kinase C is involved in the regulation and desensitisation of this receptor. We investigated this possibility by measuring angiotensin II induced Ca2+ transients in cultures of neonatal rat cardiac fibroblasts which express predominantly the angiotensin AT1A receptor. Stimulating or inhibiting protein kinase C activity had no effect on angiotensin II stimulated Ca2+ transients. In addition, in situ and in vitro kinase assays revealed that a peptide, corresponding to the region of the angiotensin AT1A receptor containing the protein kinase C sites, was a poor substrate for protein kinase C. Thus, a heterologous desensitising role for this kinase on angiotensin AT1A receptors in these fibroblasts appears unlikely.

The role of the renin-angiotensin system in the pathophysiology of cardiac remodeling.

Cardiac hypertrophy of diverse etiologies is associated with two remodeling events: an increase in cardiac muscle mass, and the abnormal accumulation of fibrillar collagen, which results in increased myocardial stiffness and eventual ventricular dysfunction. Clinical and animal studies have implicated angiotensin II (A II) as a growth promoter of both cardiac myocytes and fibroblasts during the cardiac remodeling that occurs with hypertension and myocardial infarction. The growth-promoting effects of A II occur, in part, independent of effects on hemodynamic load. Tissue culture studies have shown that cardiac myocytes and fibroblasts are targets for the actions of A II. In these cells. A II activates phospholipases C, D, and A2, leading in turn to the activation of multiple, conventional second-messenger pathways. By an undefined process. A II also increases the tyrosine phosphorylation of cytosolic proteins, and activates the STAT family of transcription factors, which may mediate an inflammatory or stress response. A II has been shown to affect gene expression of cultured cardiac myocytes and fibroblasts, induce either cellular hyperplasia or hypertrophy, and increase expression of other growth factors. Cardiac fibroblasts have been shown to respond to A II with increased expression of integrins and the extracellular matrix proteins, collagen and fibronectin. Recently, stretch of cardiac myocytes was shown to induce hypertrophy, through an autocrine release of A II. All of the aforementioned actions of A II are mediated by the AT1 receptor.

Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and beta-adrenergic stimulation.

We previously demonstrated the presence of components for a renin-angiotensin system in fibroblasts cultured from neonatal rat ventricles, the regulation of expression of which has not been studied. Since glucocorticoids and beta-adrenergic stimuli have been implicated in cardiac hypertrophy, and function as regulators of the circulating renin-angiotensin system, we examined the effects of dexamethasone and isoproterenol on angiotensinogen mRNA levels and protein secretion in cultured neonatal rat cardiac fibroblasts. Treatment of cardiac fibroblasts for 8 h with 10 micromol/l isoproterenol or 100 nmol/l dexamethasone increased angiotensinogen mRNA levels by 246 +/- 7% and 1406 +/- 207%, respectively. Over 24 h, dexamethasone and isoproterenol increased angiotensinogen secretion by 148 +/- 32% and 123 +/- 26%, respectively. Angiotensin II, which has been reported to be a positive regulator of angiotensinogen synthesis and secretion in liver, markedly attenuated the effects of dexamethasone and isoproterenol on angiotensinogen mRNA expression and secretion. In the presence of 1 micromol/l angiotensin II, the stimulation in angiotensinogen secretion observed with dexamethasone and isoproterenol was decreased by 62% and 76%, respectively. The negative feedback of angiotensin II on angiotensinogen expression was primarily mediated through the type one angiotensin II (AT1) receptor (IC50 = 0.30 +/- 0.02 nmol/l). In summary, results from this study demonstrate that angiotensinogen mRNA levels and protein secretion in cardiac fibroblasts are positively regulated by glucocorticoid and beta-adrenergic stimulation. In addition, angiotensinogen production by cardiac fibroblasts is under negative feedback control of angiotensin II.

Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system.

The renin-angiotensin system has a varied role in the regulation of cardiac function, ranging from early receptor-mediated effects such as second messenger generation, to more delayed responses such as protein synthesis and cell growth. Clinically, the importance of the RAS in cardiovascular disease is becoming increasingly evident with the use of ACE inhibitors in treating various pathological processes. With evidence for the existence of a local RAS in the heart, the molecular and biochemical regulation of this system requires investigation. Much additional work needs to be directed toward elucidating the mechanisms by which the AII-receptor couples to cardiac growth, how the local RAS is regulated, and the nature of controls that modulate cardiac production and actions of this peptide. Increased understanding of the mechanisms by which AII actions are affected in cardiac tissue will likely lead to enhanced therapeutic modalities for the treatment of pathological cardiovascular conditions in which the RAS plays an integral role.

Renal sugar transport in the winter flounder. VI. Reabsorption of D-mannose.

The transport of D-mannose (Man) in flounder kidney was studied using renal clearance techniques in vivo and brush border membrane (BBM) vesicles in vitro. At plasma concentrations of 50-100 microM Man, the winter flounder (Pseudopleuronectes americanus) reabsorbed up to 70% of the filtered sugar. Man phosphates, but not free Man, accumulated in renal cells. Reabsorption of Man was reduced by phlorizin, D-glucose, methyl-alpha-D-glucoside, methyl-alpha-D-mannoside, and 2-deoxy-D-glucose. In BBM vesicles a Na+-dependent, phlorizin-sensitive overshoot in Man uptake (10 microM) was seen. Na+-dependent Man uptake was saturable, with an apparent Km of 127 microM. The transport properties for Man were identical in BBM vesicles from the winter flounder and southern flounder (Paralichthys lethostigma). The transport specificity was determined by cis-inhibition and trans-stimulation experiments with BBM. Glucose, galactose, 1,5-anhydro-D-mannitol (i.e., 1-deoxymannose), 2-deoxy-2-fluoro-D-glucose, and methyl-alpha-mannoside were shown to share the carrier-mediating mannose transport. 2-Deoxyglucose, methyl-alpha-2-deoxy-D-glucoside, and both the isomers (alpha and beta) of methyl-D-glucoside did not. In contrast, alpha-methyl-D-glucoside inhibited D-glucose transport both in vivo and in BBM vesicles. It is concluded that Man reabsorption in the flounder occurs via a Na+-cotransport system that also handles glucose but that differs from the glucose/methyl-alpha-D-glucoside reabsorptive pathway in that 1) an oxygen on C-1 is not required, and 2) an axial configuration for -OH on C-2 (C1 conformation) is readily accommodated.

Angiotensin II signalling pathways in cardiac fibroblasts: conventional versus novel mechanisms in mediating cardiac growth and function.

Angiotensin II has been demonstrated to be involved in the regulation of cellular growth of several tissues in response to developmental, physiological, and pathophysiological processes. Angiotensin II has been implicated in the developmental growth of the left ventricle in the neonate and remodeling of the heart following chronic hypertension and myocardial infarction. The inhibition of DNA synthesis and collagen deposition in myocardial interstitium following myocardial infarction by angiotensin converting enzyme inhibitor, suggests that angiotensin II mediates interstitial and perivascular fibrobrosis by preventing fibroblast proliferation. In the past, little attention was focused on the identity and functional roles of cardiac fibroblasts. Recent in vitro studies utilizing cultured cardiac fibroblasts demonstrate that angiotensin II, acting via the AT1 receptor, initiates intracellular signalling pathways in common with those of peptide growth factors. Below, we describe growth-related aspects of cardiac fibroblasts with respect to angiotensin II receptors, conventional and novel signal transduction systems, secretion of extracellular matrix proteins and growth factors, and localization of renin-angiotensin system components.

Propionate induces cell swelling and K+ accumulation in shark rectal gland.

Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by 86Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes.

Involvement of protein kianse C and Ca2+ in angiotensin II-induced mitogenesis of cardiac fibroblasts.

Angiotensin (ANG) II has been previously shown to stimulate proliferation of neonatal rat cardiac fibroblasts via AT1 receptors. Here we conducted studies to assess involvement in this process of two second messengers linked to AT1 receptors, protein kinase C (PKC) and Ca2+. Several findings argue against a dominant role for PKC in ANG II-induced mitogenesis: 1) [Sar1]ANG II, which produced a modest, transient increase in PKC activity, was equally effective in inducing thymidine incorporation into DNA in PKC-depleted cells, whereas the effect of platelet-derived growth factor (PDGF)-BB on thymidine incorporation was reduced to the level observed with [Sar1]ANG II; 2) phorbol 12-myristate 13-acetate (PMA), a potent PKC stimulator, was ineffective in stimulating thymidine incorporation; and 3) PKC downregulation or the highly specific PKC inhibitor, compound 3, eliminated PMA-induced mitogen-activated protein (MAP) kinase activity but did not affect comparable increases induced by [Sar1]ANG II or PDGF-BB. Increased intracellular Ca2+ may be sufficient to account for [Sar1]ANG II-induced MAP kinase activity because ionomycin also increased MAP kinase activity and chelation of intracellular Ca2+ eliminated [Sar1]ANG II-induced activity in PKC-depleted fibroblasts. However, Ca2+ chelation did not prevent [Sar1]ANG II-induced MAP kinase activity in non-PKC-depleted fibroblasts. Thus ANG II can activate MAP kinase in cardiac fibroblasts by either Ca(2+)- or PKC-dependent pathways, and whereas the full effect of PDGF-BB on thymidine incorporation and cell proliferation requires a phorbol ester-sensitive PKC, the hyperplastic growth effect of ANG II does not.

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II is mitogenic in neonatal rat cardiac fibroblasts.

Angiotensin II has been reported to be a hormonal stimulus of cardiac growth, a response that may involve myocyte hypertrophy as well as growth of nonmyocytes. This study was designed to determine whether neonatal rat cardiac fibroblasts have an angiotensin II receptor that is coupled with hypertrophic and/or proliferative growth. Competitive radioligand binding studies showed that cardiac fibroblasts have a single class of high-affinity (IC50, 1.0 nM) angiotensin II binding sites (Bmax, 778 fmol/mg protein) that are sensitive to the competitive nonpeptide AT1 receptor antagonist losartan (IC50, 13 nM). Other angiotensin peptides competed for [125I]angiotensin II binding in the following rank order: angiotensin II > angiotensin III > angiotensin I > > [des-Asp1-des-Arg2]angiotensin II. A nonhydrolyzable analogue of guanosine triphosphate increased the dissociation rate of bound [125I]angiotensin II and decreased hormone binding to the receptor at equilibrium. The angiotensin II receptor was coupled with increases in intracellular calcium. Incorporation of precursors into protein, DNA, and RNA in response to angiotensin II was determined. In serum-deprived cultures, a 24-hour exposure to 1 microM [Sar1]angiotensin II increased rates of phenylalanine, thymidine, and uridine incorporation by 58%, 103%, and 118%, respectively. These increases were blocked by the noncompetitive AT1 receptor antagonist EXP3174. After 48 hours, [Sar1]angiotensin II increased total protein and DNA of cardiac fibroblasts by 23% and 15%, respectively, with no change in the protein/DNA ratio. [Sar1]Angiotensin II increased cell number by 138% after a 24-hour exposure, without affecting cell area. In summary, cardiac fibroblasts have G protein-linked AT1 receptors that are coupled with proliferative growth. These results suggest that angiotensin II-induced cardiac hypertrophy is, in part, secondary to stimulated increases in nonmyocyte cellular growth.