ANG II-induced cell proliferation is dually mediated by c-Src/Yes/Fyn-regulated ERK1/2 activation in the cytoplasm and PKCzeta-controlled ERK1/2 activity within the nucleus

High-affinity binding of angiotensin II (ANG II) to the ANG II type 1 receptor (AT₁R) results in the activation of ERK1/2 mitogen-activated protein kinases (MAPK). However, the precise mechanism of ANG II-induced ERK1/2 activation has not been fully characterized. Here, we investigated the signaling events leading to ANG II-induced ERK1/2 activation using a c-Src/Yes/Fyn tyrosine kinase-deficient mouse embryonic fibroblast (MEF) cell line stably transfected with the AT₁R (SYF/AT₁). ERK1/2 activation was reduced by 50% within these cells compared with wild-type controls (WT/AT₁). The remaining 50% of intracellular ERK1/2 activation was dependent upon heterotrimeric G protein and protein kinase C zeta (PKC) activation. Therefore, ANG II-induced ERK1/2 activation occurs via two independent mechanisms. We next investigated whether a loss of either c-Src/Yes/Fyn or PKC signaling affected ERK1/2 nuclear translocation and cell proliferation in response to ANG II. ANG II-induced cell proliferation was markedly reduced in SYF/AT₁ cells compared with WT/AT₁ cells (P < 0.01), but interestingly, ERK2 nuclear translocation was normal. ANG II-induced nuclear translocation of ERK2 was blocked via pretreatment of WT/AT₁ cells with a PKC pseudosubstrate. ANG II-induced cell proliferation was significantly reduced in PKC pseudosubstrate-treated WT/AT₁ cells (P < 0.01) and was completely blocked in SYF/AT₁ cells treated with this same compound. Thus ANG II-induced cell proliferation appears to be regulated by both ERK1/2-driven nuclear and cytoplasmic events. In response to ANG II, the ability of ERK1/2 to remain within the cytoplasm or translocate into the nucleus is controlled by c-Src/Yes/Fyn or heterotrimeric G protein/PKC signaling, respectively. Src family tyrosine kinases; angiotensin II     

ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling

ANG II type 1 (AT₁) receptors respond to sustained exposure to ANG II byundergoing downregulation of absolute receptor numbers. It has beenassumed previously that downregulation involves endocytosis. Thepresent study hypothesized that AT₁ receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT₁ receptors with carboxy-terminal deletionsinternalized <5% of radioligand compared with 65% for wild-typeAT₁ receptors. The truncated AT₁ receptorsretained the ability to undergo downregulation. These data suggest theexistence of an alternative pathway to AT₁ receptordegradation that does not require endocytosis, per se. Point mutationsin either the second transmembrane region or second intracellular loopimpaired G protein (G_q) coupling. These receptors exhibiteda biphasic pattern of downregulation. The earliest phase ofdownregulation (0-2 h) was independent of coupling toG_q, but no additional downregulation was observed after2 h of ANG II exposure in the receptors with impaired coupling toG_q. These data suggest that coupling to G_q isrequired for the later phase (2-24 h) of AT₁ receptor downregulation.

     

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G alpha i3/Ras/Raf/MAPK pathway

We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G_i3 dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT₂ antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G_i3, Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT₂-dependent increase in eNOS expression in PAECs. Src; mitogen-activated protein kinase     

Cleavage of the angiotensin II type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment

Our published studies show that the distribution of the ANG II type 1 (AT₁) receptor (AT₁R), expressed as a enhanced yellow fluorescent fusion (YFP) protein (AT₁R/EYFP), is altered upon cellular treatment with ANG II or coexpression with intracellular ANG II. AT₁R accumulates in nuclei of cells only in the presence of ANG II. Several transmembrane receptors are known to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. The present study was designed to determine whether the AT₁R is cleaved before nuclear transport. A plasmid encoding a rat AT₁R labeled at the amino terminus with enhanced cyan fluorescent protein (CFP) and at the carboxy terminus with EYFP was employed. Image analyses of this protein in COS-7 cells, CCF-STTG1 glial cells, and A10 vascular smooth muscle cells show the two fluorescent moieties to be largely spatially colocalized in untreated cells. ANG II treatment, however, leads to a separation of the fluorescent moieties with yellow fluorescence accumulating in more than 30% of cellular nuclei. Immunoblot analyses of extracts and conditioned media from transfected cells indicate that the CFP domain fused to the extracellular amino-terminal AT₁R domain is cleaved from the membrane and that the YFP domain, together with the intracellular cytoplasmic carboxy terminus of the AT₁R, is also cleaved from the membrane-bound receptor. The carboxy terminus of the AT₁R is essential for cleavage; cleavage does not occur in protein deleted with respect to this region. Overexpressed native AT₁R (nonfusion) is also cleaved; the intracellular 6-kDa cytoplasmic domain product accumulates to a significantly higher level with ANG II treatment. nuclear angiotensin II type 1 receptor; intracrine; intracellular     

A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption

Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (I_sc) by 15–25%, whereas the addition of ATP to the apical bathing solution decreased I_sc by 40–60%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive I_sc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive I_sc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in I_sc, but did not alter the EGF- or ATP-induced inhibition of I_sc. The DBHQ-mediated decrease in I_sc was due to inhibition of basolateral Na⁺-K⁺-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na⁺ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel     

Intracellular angiotensin II induces cell proliferation independent of AT1 receptor

We recently reported intracrine effects of angiotensin II (ANG II) on cardiac myocyte growth and hypertrophy that were not inhibited by the ANG II type 1 receptor (AT₁) antagonist, losartan. To further determine the role of AT₁ in intracrine effects, we studied the effect of intracellular ANG II (iANG II) on cell proliferation in native Chinese hamster ovary (CHO) cells and those stably transfected with AT₁ receptor (CHO-AT₁). CHO-AT₁, but not CHO cells, showed enhanced proliferation following exposure to extracellular ANG II (eANG II). However, when transiently transfected with an iANG II expression vector, both cell types showed significantly enhanced proliferation, compared with those transfected with a scrambled peptide. Losartan blocked eANG II-induced cell proliferation, but not that induced by iANG II. To further confirm these findings, CHO and CHO-AT₁ cells were stably transfected for iANG II expression (CHO-iA and CHO-AT₁-iA, respectively). Cells grown in serum-free medium were counted every 24 h, up to 72 h. CHO-iA and CHO-AT₁-iA cells showed a steeper growth curve compared with CHO and CHO-AT₁, respectively. These observations were confirmed by Wst-1 assay. The AT₁ receptor antagonists losartan, valsartan, telmisartan, and candesartan did not attenuate the faster growth rate of CHO-iA and CHO-AT₁-iA cells. eANG II showed an additional growth effect in CHO-AT₁-iA cells, which could be selectively blocked by losartan. These data demonstrate that intracrine ANG II can act independent of AT₁ receptors and suggest novel intracellular mechanisms of action for ANG II. renin-angiotensin system; angiotensinogen; peptide hormones; nuclear signaling; intracrine     

Effect of chronically elevated CO2 on CA1 neuronal excitability

To study the effect of chronically elevated CO₂ on the excitability and function of neurons, we exposed mice to 7.5–8% CO₂ for 2 wk (starting at 2 days of age) and examined the properties of freshly dissociated hippocampal neurons. Neurons from control mice (CON) and from mice exposed to chronically elevated CO₂ had similar resting membrane potentials and input resistances. CO₂-exposed neurons, however, had a lower rheobase and a higher Na⁺ current density (580 ± 73 pA/pF; n = 27 neurons studied) than did CON neurons (280 ± 51 pA/pF, n = 34; P < 0.01). In addition, the conductance-voltage curve was shifted in a more negative direction in CO₂-exposed than in CON neurons (midpoint of the curve was –46 ± 3 mV for CO₂ exposed and –34 ± 3 mV for CON, P < 0.01), while the steady-state inactivation curve was shifted in a more positive direction in CO₂-exposed than in CON neurons (midpoint of the curve was –59 ± 2 mV for CO₂ exposed and –68 ± 3 mV for CON, P < 0.01). The time constant for deactivation at –100 mV was much smaller in CO₂-exposed than in CON neurons (0.8 ± 0.1 ms for CO₂ exposed and 1.9 ± 0.3 ms for CON, P < 0.01). Immunoblotting for Na⁺ channel proteins (subtypes I, II, and III) was performed on the hippocampus. Our data indicate that Na⁺ channel subtype I, rather than subtype II or III, was significantly increased (43%, n = 4; P < 0.05) in the hippocampi of CO₂-exposed mice. We conclude that in mice exposed to elevated CO₂, 1) increased neuronal excitability is due to alterations in Na⁺ current and Na⁺ channel characteristics, and 2) the upregulation of Na⁺ channel subtype I contributes, at least in part, to the increase in Na⁺ current density. sodium ion channels; oxygen deprivation     

Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system

The renin-angiotensin system is a central component of the physiological and pathological responses of cardiovascular system. Its primary effector hormone, angiotensin II (ANG II), not only mediates immediate physiological effects of vasoconstriction and blood pressure regulation, but is also implicated in inflammation, endothelial dysfunction, atherosclerosis, hypertension, and congestive heart failure. The myriad effects of ANG II depend on time (acute vs. chronic) and on the cells/tissues upon which it acts. In addition to inducing G protein- and non-G protein-related signaling pathways, ANG II, via AT₁ receptors, carries out its functions via MAP kinases (ERK 1/2, JNK, p38MAPK), receptor tyrosine kinases [PDGF, EGFR, insulin receptor], and nonreceptor tyrosine kinases [Src, JAK/STAT, focal adhesion kinase (FAK)]. AT₁R-mediated NAD(P)H oxidase activation leads to generation of reactive oxygen species, widely implicated in vascular inflammation and fibrosis. ANG II also promotes the association of scaffolding proteins, such as paxillin, talin, and p130Cas, leading to focal adhesion and extracellular matrix formation. These signaling cascades lead to contraction, smooth muscle cell growth, hypertrophy, and cell migration, events that contribute to normal vascular function, and to disease progression. This review focuses on the structure and function of AT₁ receptors and the major signaling mechanisms by which angiotensin influences cardiovascular physiology and pathology. vascular smooth muscle; NAD(P)H oxidase; tyrosine and nontyrosine receptor kinases; endothelial dysfunction; vascular disease     

Extracellular pressure stimulates macrophage phagocytosis by inhibiting a pathway involving FAK and ERK

We hypothesized that changes in extracellular pressure during inflammation or infection regulate macrophage phagocytosis through modulating the focal adhesion kinase (FAK)-ERK pathway. Undifferentiated (monocyte-like) or PMA-differentiated (macrophage-like) THP-1 cells were incubated at 37°C with serum-opsonized latex beads under ambient or 20-mmHg increased pressure. Pressure did not affect monocyte phagocytosis but significantly increased macrophage phagocytosis (29.9 ± 1.8 vs. 42.0 ± 1.6%, n = 9, P < 0.001). THP-1 macrophages constitutively expressed activated FAK, ERK, and Src. Exposure of macrophages to pressure decreased ERK and FAK-Y397 phosphorylation (77.6 ± 7.9%, n = 7, P < 0.05) but did not alter FAK-Y576 or Src phosphorylation. FAK small interfering RNA (SiRNA) reduced FAK expression by >75% and the basal amount of phosphorylated FAK by 25% and significantly increased basal macrophage phagocytosis (P < 0.05). Pressure inhibited FAK-Y397 phosphorylation in mock-transfected or scrambled SiRNA-transfected macrophages, but phosphorylated FAK was not significantly reduced further by pressure in cells transfected with FAK SiRNA. Pressure increased phagocytosis in all three groups. However, FAK-SiRNA-transfected cells exhibited only 40% of the pressure effect on phagocytosis observed in scrambled SiRNA-transfected cells so that phagocytosis inversely paralleled FAK activation. PD-98059 (50 µM), an ERK activation inhibitor, increased basal phagocytosis (26.9 ± 1.8 vs. 31.7 ± 1.1%, n = 15, P < 0.05), but pressure did not further increase phagocytosis in PD-98059-treated cells. Pressure also inhibited ERK activation after mock transfection or transfection with scrambled SiRNA, but transfection of FAK SiRNA abolished ERK inhibition by pressure. Pressure did not increase phagocytosis in MonoMac-1 cells that do not express FAK. Increased extracellular pressure during infection or inflammation enhances macrophage phagocytosis by inhibiting FAK and, consequently, decreasing ERK activation. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction     

Effect of caveolin-1 scaffolding peptide and 17 -estradiol on intracellular Ca2+ kinetics evoked by angiotensin II in human vascular smooth muscle cells

Caveolae are identifiable plasma membrane invaginations. The main structural proteins of caveolae are the caveolins. There are three caveolins expressed in mammals, designated Cav-1, Cav-2, and Cav-3. It has been postulated that Cav-1 acts as a scaffold protein for signaling proteins; these include ion channels, enzymes, and other ligand receptors like membrane-associated estrogen receptor (ER) or ERβ. Caveolae-associated membrane proteins are involved in regulating some of the rapid estrogenic effects of 17β-estradiol. One important system related to the activity of ER and caveolae is the renin-angiotensin system. Angiotensin II (ANG II) has numerous actions in vascular smooth muscle, including modulation of vasomotor tone, cell growth, apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt activation, and others. Many proteins associated with caveolae are in close relation with the scaffolding domain of Cav-1 (82–101 amino acid residues). It has been proposed that this peptide may acts as a kinase inhibitor. Therefore, to explore the ability of Cav-1 scaffolding peptide (CSP-1) to regulate ANG II function and analyze the relationship between ER and ANG II type 1 and 2 (AT₁ and AT₂) receptors, we decided to study the effects of CSP-1 on ANG II-induced intracellular Ca²⁺ kinetics and the effect of 17β-estradiol on this modulation using human smooth muscle cells in culture, intracellular Ca²⁺ concentration measurements, immuno- and double-immunocytochemistry confocal analysis of receptor expression, immunoblot analysis, and immunocoprecipitation assays to demonstrate coexpression. We hypothesized that CSP-1 inhibits ANG II-mediated increases in intracellular Ca²⁺ concentrations by interfering with intracellular signaling including the PI3K/Akt pathway. We also hypothesize that AT₂ receptors associate with Cav-1. Our results show that there is a close association of AT₁, AT₂, and ER with Cav-1 in human arterial smooth muscle cells in culture. CSP-1 inhibits ANG II-induced intracellular signaling. estrogen receptors; angiotensin type 1 and 2 receptors; phosphatidylinositol 3-kinase; intracellular signaling; tissue culture; angiotensin receptors     

Atrial natriuretic peptide levels and plasma volume contraction in acute alveolar hypoxia

Albert, T. S. E., V. L. Tucker, and E. M. Renkin.Atrial natriuretic peptide levels and plasma volume contraction in acute alveolar hypoxia. J. Appl.Physiol. 82(1): 102-110, 1997.Arterial oxygentensions (Pa_O2), atrial natriureticpeptide (ANP) concentrations, and circulating plasma volumes (PV) weremeasured in anesthetized rats ventilated with room air or 15, 10, or8% O₂(n = 5-7). After 10 min ofventilation, Pa_O2 values were 80 ± 3, 46 ± 1, 32 ± 1, and 35 ± 1 Torrand plasma immunoreactive ANP (irANP) levels were 211 ± 29, 229 ± 28, 911 ± 205, and 4,374 ± 961 pg/ml, respectively. AtPa_O2 40 Torr, irANP responses weremore closely related to inspiredO₂(P = 0.014) than toPa_O2 (P = 0.168). PV was 36.3 ± 0.5 µl/g in controls but 8.5 and9.9% lower (P  0.05) for10 and 8% O₂, respectively.Proportional increases in hematocrit were observed in animals withreduced PV; however, plasma protein concentrations were not differentfrom control. Between 10 and 50 min of hypoxia, small increases (+40%)in irANP occurred in 15% O₂;however, there was no further change in PV, hematocrit, plasma protein,or irANP levels in the lower O₂groups. Urine output tended to fall during hypoxia but was notsignificantly different among groups. These findings are compatiblewith a role for ANP in mediating PV contraction during acute alveolarhypoxia.

     

Prostaglandin production contributes to exercise-induced vasodilation in heart failure

Lang, Chim C., Don B. Chomsky, Javed Butler, Shiv Kapoor,and John R. Wilson. Prostaglandin production contributes toexercise-induced vasodilation in heart failure. J. Appl. Physiol. 83(6): 1933-1940, 1997.Endothelial release of prostaglandins may contribute toexercise-induced skeletal muscle arteriolar vasodilation in patientswith heart failure. To test this hypothesis, we examined the effect ofindomethacin on leg circulation and metabolism in eight chronic heartfailure patients, aged 55 ± 4 yr. Central hemodynamics and legblood flow, determined by thermodilution, and leg metabolic parameterswere measured during maximum treadmill exercise before and 2 h afteroral administration of indomethacin (75 mg). Leg release of6-ketoprostaglandin F₁ was alsomeasured. During control exercise, leg blood flow increased from 0.34 ± 0.03 to 1.99 ± 0.19 l/min(P < 0.001), legO₂ consumption from 13.6 ± 1.8 to 164.5 ± 16.2 ml/min (P < 0.001), and leg prostanoid release from 54.1 ± 8.5 to267.4 ± 35.8 pg/min (P < 0.001).Indomethacin suppressed release of prostaglandinF₁(P < 0.001) throughout exercise anddecreased leg blood flow during exercise(P < 0.05). This was associated witha corresponding decrease in leg O₂ consumption (P < 0.05) and a higher level offemoral venous lactate at peak exercise(P < 0.01). These data suggest thatrelease of vasodilatory prostaglandins contributes to skeletal musclearteriolar vasodilation in patients with heart failure.

     

Effect of crystalloid administration on oxygen extraction in endotoxemic pigs

We asked whethercrystalloid administration improves tissue oxygen extraction inendotoxicosis. Four groups of anesthetized pigs(n = 8/group) received either normalsaline infusion or no saline and either endotoxin or no endotoxin. Wemeasured whole body (WB) and gut oxygen delivery and consumption duringhemorrhage to determine the critical oxygen extraction ratio(ER_O2 crit). Just after onset of ischemia (critical oxygen delivery rate), gut was removed for determination of area fraction of interstitial edema and capillary hematocrit. Radiolabeled microspheres were used todetermine erythrocyte transit time for the gut. Endotoxin decreased WBER_O2 crit(0.82 ± 0.06 to 0.55 ± 0.08, P < 0.05) and gutER_O2 crit(0.77 ± 0.07 to 0.52 ± 0.06, P < 0.05). Unexpectedly, saline administration also decreased WBER_O2 crit (0.82 ± 0.06 to 0.62 ± 0.08, P < 0.05) and gutER_O2 crit (0.77 ± 0.07 to 0.67 ± 0.06, P < 0.05) in nonendotoxin pigs. Saline administration increased thearea fraction of interstitial space (P < 0.05) and resulted in arterial hemodilution(P < 0.05) but not capillaryhemodilution (P > 0.05). Salineincreased the relative dispersion of erythrocyte transit times from0.33 ± 0.08 to 0.72 ± 0.53 (P < 0.05). Thus saline administration impairs tissue oxygen extractionpossibly by increasing interstitial edema or increasing heterogeneityof microvascular erythrocyte transit times.

     

Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells

Angiotensin II(ANG II) produces vasoconstriction by a direct action on smooth musclecells via AT₁ receptors. Thesereceptors are also present in the endothelium, but their function ispoorly understood. This study was therefore undertaken to determinewhether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by theaccumulation of nitrite and nitrate, was enhanced by10⁷ M ANG II. Thebiological activity of the NO released by ANG II action was evaluatedby measuring its guanylate cyclase-stimulating activity in smoothmuscle cells. The guanosine 3',5'-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased byexposure of supernatant from ANG II-stimulated endothelial cells. Theseeffects resulted from the activation of NO synthase, as they wereinhibited by the L-arginineanalogs. These ANG II actions were mediated by theAT₁ receptor, as shown by theirinhibition by the AT₁ antagonistlosartan. The cGMP production by reporter cells was inhibited by thecalmodulin antagonist W-7, suggesting that ANG II activates endothelialcalmodulin-dependent NO synthase. This hypothesis is also supported bythe increase of intracellular free calcium induced by ANG II inendothelial cells. ANG II also stimulated luminol-enhancedchemiluminescence in endothelial cells. This effect was inhibited byN-monomethyl-L-arginine andsuperoxide dismutase, suggesting that this luminol-enhancedchemiluminescence reflected an increase in peroxynitrite production.Thus ANG II stimulates NO release from macrovascular endothelium, whichmay modulate the direct vasoconstrictor effect of ANG II on smoothmuscle cells. However, this beneficial effect may be counteracted bythe simultaneous production of peroxynitrite, which could contribute toseveral pathological processes in the vascular wall.

     

Esophageal temperature threshold for sweating decreases before ovulation in premenopausal women

The purpose ofthis study was to test the hypothesis that regulated body temperatureis decreased in the preovulatory phase in eumenorrheic women. Six womenwere studied in both the preovulatory phase (Preov-2;days 9-12), which was 1-2days before predicted ovulation when 17-estradiol(E₂) was estimated to peak, andin the follicular phase (F; days2-6). The subjects walked on a treadmill (~225W · m²)in a warm chamber (ambient temperature = 30°C; dew-pointtemperature = 11.5°C) while heavily clothed.E₂, esophageal temperature(T_es), local skin temperatures,and local sweating rate were measured. The estimate of when theE₂ surge would occur was correctfor four of six subjects. In these four subjects,E₂ increased(P  0.05) from 42.0 ± 24.5 pg/mlduring F to 123.2 ± 31.3 pg/ml during Preov-2. RestingT_es was 37.02 ± 0.20°Cduring F and 36.76 ± 0.28°C during Preov-2(P  0.05). TheT_es threshold for sweating wasdecreased (P  0.05) from 36.88 ± 0.27°C during F to 36.64 ± 0.35°C during Preov-2. Both meanskin and mean body temperatures were decreased during rest in Preov-2group. The hypothesis that regulated body temperature is decreasedduring the preovulatory phase is supported.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells

In myogenic C₂C₁₂ cells, 5 mM creatine increased the incorporation of labeled [³⁵S]methionine into sarcoplasmic (+20%, P < 0.05) and myofibrillar proteins (+50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+40%, P < 0.001). Expression of myosin heavy chain type II (+1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and -alanine did not mimic the effect of creatine, ruling out an osmolarity-dependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70^s6k) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70^s6k and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70^s6k (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (–50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (–55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70^s6k pathways in the enhanced differentiation induced by creatine in C₂C₁₂ cells. protein synthesis; insulin-like growth factor; mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2; 70-kDa ribosomal S6 protein kinase     

Linear Relations between Carbon Dioxide Concentration and Rate of Development Towards Flowering in Sorghum, Cowpea and Soyabean

Negative linear relations were detected (P < 0·005)between the rate of progress from sowing to panicle initiationand CO₂ concentration (210-720 µmol CO₂ mol^-1 air) fortwo genotypes of sorghum [Sorghum bicolor (L.) Moench]. Relationsbetween CO₂ concentration and the rate of progress from sowingto first flowering were also negative in soyabean [Glycine max(L.) Merrill] (P < 0·025), but positive in cowpea[Vigna unguiculata (L.) Walp.] (P < 0·025), albeitthat in both grain legumes sensitivity was much less than insorghum. Thus CO₂ elevation does not delay flowering in allshort-day species. The considerable effect of CO₂ concentrationon times to panicle initiation resulted in large differencesamong the sorghum plants at this developmental stage; with increasein CO₂ concentration, plants were taller with slightly moreleaves and more pronounced apical extension. At the same timeafter sowing however, sorghum plants were heavier (P < 0·05)at 210 than at 360 µmol CO₂ mol^-1 air. In contrast, relationsbetween the dry masses of the soyabean and cowpea plants andCO₂ concentration were positive and curvilinear (P < 0·05).It is suggested that the impact of global environmental changecould be severe for sorghum production in the semi-arid tropics.Copyright1995, 1999 Academic Press Sorghum bicolor (L.) Moench., sorghum, Glycine max (L.) Merrill, soyabean, Vigna unguiculata (L.) Walp., cowpea, development, flowering, CO₂, dry matter accumulation, environmental change     

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells

Angiotensin II(ANG II) has long been known for its pressor and growth-promotingeffects, which are both mediated by theAT₁ receptor. By contrast, theAT₂ receptor has recently beenreported to mediate inhibition of proliferation through as yetundefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks thecells in G₁ phase. Consistent withthis, ANG II inhibits cyclin D₁ expression and cyclinD₁-associated kinase activity. Theantimitogenic effect of ANG II is partly mimicked by theAT₂-selective agonist CGP-42112.It is also blocked partly and in an additive fashion by theAT₁- andAT₂-selective antagonists losartanand PD-123319, indicating the contribution of both receptor subtypes tothis response. AT₁-dependentantiproliferation is selectively blocked by the cyclooxygenaseinhibitor indomethacin and restored by prostaglandin E₂, whereasAT₂-receptor-mediated inhibitionof growth is suppressed by the tyrosine phosphatase inhibitorsorthovanadate and bpV(pic). Both pathways are, however,pertussis toxin sensitive. We hypothesize that, in fasciculatacells, the AT₁ receptor inhibitsbFGF-induced proliferation by stimulating prostaglandin synthesis,whereas the AT₂ receptor mediatesits effect through a pathway that requires protein tyrosine phosphataseactivation.

     

Influence of hydrostatic pressure gradients on regulation of plasma volume after exercise

The impact of posture on the immediate recoveryof intravascular fluid and protein after intense exercise wasdetermined in 14 volunteers. Forces which govern fluid and proteinmovement in muscle interstitial fluid pressure(P_ISF), interstitial colloid osmotic pressure (COP_i), andplasma colloid osmotic pressure(COP_p) were measured before andafter exercise in the supine or upright position. During exercise,plasma volume (PV) decreased by 5.7 ± 0.7 and 7.0 ± 0.5 ml/kgbody weight in the supine and upright posture, respectively. Duringrecovery, PV returned to its baseline value within 30 min regardless ofposture. PV fell below this level by 60 and 120 min in the supine andupright posture, respectively (P < 0.05). Maintenance of PV in the upright position was associated with adecrease in systolic blood pressure, an increase inCOP_p (from 25 ± 1 to 27 ± 1 mmHg; P < 0.05), and an increasein P_ISF (from 5 ± 1 to 6 ± 2 mmHg), whereas COP_i wasunchanged. Increased P_ISFindicates that the hydrostatic pressure gradient favors fluid movementinto the vascular space. However, retention of the recaptured fluid inthe plasma is promoted only in the upright posture because of increasedCOP_p.