inositol-1-4-5-trisphosphate and Hypertension

There is universal acceptance of the existence of rapid, non-genomic effects of aldosterone, although their physiological relevance and potential importance in hypertension are not yet clear. What has emerged over the year under review is that at least some of such rapid non-genomic effects of aldosterone may be mediated by the activation of the classical intracellular mineralocorticoid receptor, rather than a putative membrane receptor. The post-receptor mechanisms of rapid aldosterone action appear variously to involve protein kinase C, calcium, cyclic adenosine 3', 5'-monophosphate and inositol 1, 4, 5-triphosphate, with downstream effects on a variety of ion pumps and channels.

Topics: Aldosterone; Animals; Blood Pressure; Calcium; Cyclic AMP; Humans; Hypertension; Inositol 1,4,5-Trisphosphate; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocardium; Protein Kinase C; Receptors, Mineralocorticoid

The second messenger inositol-1,4,5-trisphosphate (InsP3) comes from two major pathways, one is initiated by a family of G protein-linked receptors and the other by receptors linked by tyrosine kinases. These separate receptors activate phospholipase C to hydrolyse phosphatidylinositol-4,5-bisphosphate to give both diacylglycerol and InsP3. The latter then mobilizes stored calcium and promotes an influx of external calcium. The alpha subunit of a newly discovered G protein (Gq) has recently been shown to stimulate the activity of PLC-beta 1. The alpha subunits of the Gq class of G proteins includes G alpha q, G alpha 11, G alpha 14, G alpha 15 and G alpha 16. The important pathologic changes in hypertension are arteriolar spasm and wall thickening. Many vasoactive substances can induce contractile response and proliferation of vascular smooth muscle cells and increase InsP3 level. However, the hypertension does not cause any persistent change in Gq.

Topics: Animals; GTP-Binding Proteins; Hypertension; Inositol 1,4,5-Trisphosphate; Signal Transduction

Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent.. Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca. Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

Topics: Animals; Atrial Fibrillation; Atrial Remodeling; Cell Communication; Echocardiography; Fibroblasts; Heart Atria; Heart Failure; Humans; Hypertension; Inositol 1,4,5-Trisphosphate; Male; Myocytes, Cardiac; Rats

Induction of heme-oxygenase (HO) is an important cellular defense mechanism against oxidative and inflammatory insults. We analyzed the effects of the HO inducer, heme-arginate, on the phospholipase C (PLC)/inositol-triphosphate (IP(3)) pathway in the mesenteric arterioles of uninephrectomized (UnX) deoxycorticosterone acetate (DOCA)-salt hypertensive rats, which is a volume-overload model characterized by elevated endothelin (ET-1) and mineralocorticoid-induced oxidative/inflammatory insults. Our study included the following groups: (A) controls [(i) surgery-free Sprague-Dawley (SD) rats, (ii) UnX-Sham, (iii) UnX-Salt (0.9% NaCl+0.2% KCl) and (iv) UnX-DOCA)]; (B) UnX-DOCA-salt hypertensive rats; (C) UnX-DOCA-salt+heme-arginate; (D) UnX-DOCA-salt+heme-arginate+chromium mesoporphyrin (CrMP), the HO inhibitor; (E) UnX-DOCA-salt+CrMP (F); SD+heme-arginate, (G) UnX-DOCA-salt+vehicle dissolving heme-arginate and CrMP and (H) normal-SD+heme-arginate. Quantitative reverse transcriptase PCR, western blot, enzyme immunoassay and spectrophotometric analyses were used. Heme-arginate enhanced mesenteric arteriole HO-1, HO activity, cyclic guanosine monophosphate (cGMP) and anti-oxidants including bilirubin, ferritin, superoxide dismutase with potentiation of the total anti-oxidant capacity. Correspondingly, oxidative/inflammatory mediators such as 8-isoprostane, nuclear-factor kappaB (NF-kappaB) and ET-1 were markedly reduced. Furthermore, heme-arginate suppressed PLC activity, attenuated IP(3) and reduced resting intracellular calcium. The effects of heme-arginate were nullified by the HO inhibitor, with aggravation of oxidative/inflammatory insults. In heme-arginate-treated SD rats, the HO system was potentiated to a lesser magnitude and the suppression of ET-1, PLC, IP(3) and NF-kappaB were less accentuated, suggesting greater selectivity of HO against the ET-1-PLC-IP(3)-NF-kappaB destructive axis in the pathological condition of mineralocorticoid-induced hypertension. Given that ET-1 stimulates PLC and IP(3), which in turn activates NF-kappaB, the concomitant reduction of ET-1, PLC, IP(3) and NF-kappaB alongside the corresponding decline of resting intracellular calcium may account for the reduction of blood pressure and attenuation of oxidative/inflammatory injury by heme-arginate.

Topics: Animals; Arginine; Arterioles; Blood Pressure; Calcium; Cyclic GMP; Desoxycorticosterone; Dinoprost; Endothelin-1; Heme; Heme Oxygenase (Decyclizing); Hypertension; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Male; Mesenteric Arteries; Mineralocorticoids; NF-kappa B; Organometallic Compounds; Oxidative Stress; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Sodium Chloride, Dietary; Type C Phospholipases; Up-Regulation

Reactive oxygen species have emerged as important molecules in cardiovascular dysfunction such as diabetes and hypertension. Recent work has shown that oxidative stress and angiotensin II signaling mutually regulate each other by multiple mechanisms and contribute to the development of hypertension. Most of the known biological actions of angiotensin II can be attributed to AT1 receptors. The present study was carried out to investigate the role of renal AT1 receptor signaling in oxidative stress-mediated hypertension. Male Sprague-Dawley rats received tap water (control) or 30 mM L-buthionine sulfoximine (BSO), an oxidant, with and without 1 mM tempol (an antioxidant) for 2 wk. Compared with control rats, BSO-treated rats exhibited increased oxidative stress and reduced antioxidant levels and developed hypertension. BSO treatment also caused increased renal proximal tubular AT1 receptor protein abundance, message levels, and ligand binding. In these rats, angiotensin II caused significantly higher accumulation of inositol trisphosphate (IP3) and phospholipase C (PLC) activation which was sensitive to blockade by AT1 but not to AT2 antagonist. Also, angiotensin II-mediated, AT1-dependent MAP kinase, Na-K-ATPase, and Na/H exchanger 3 activation was higher in BSO-treated rats than in control rats. Tempol supplementation of BSO-treated rats restored redox status, normalized AT1 receptor expression, and decreased blood pressure. Tempol also normalized the angiotensin II-mediated, AT1-dependent IP3 accumulation and PLC, MAP kinase, Na-K-ATPase, and Na/H exchanger 3 stimulation. These data suggest that oxidative stress leads to AT1 receptor upregulation, which in turn causes overstimulation of sodium transporters and subsequently contributes to sodium retention and hypertension. Tempol, while reducing oxidative stress, normalizes AT1 receptor signaling and decreases blood pressure.

Topics: Angiotensin II; Animals; Buthionine Sulfoximine; Enzyme Activation; Hypertension; Inositol 1,4,5-Trisphosphate; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Oxidative Stress; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Sodium-Potassium-Exchanging ATPase; Type C Phospholipases; Up-Regulation

Phospholipase C (PLC) and the small G protein RhoA are vital elements for the contraction of vascular smooth muscle cells. The available evidence points to altered PLC-delta1 activity as an element determining enhanced vascular tone in hypertension; however, the factor(s) responsible for increased PLC activity remains unknown. There is the data indicating that RhoA inhibits PLC-delta1 and factors downmodulating RhoA activate phospholipase. In the present study, we explore an impact of a newly identified human ARHGAP6 protein possessing GTPase stimulating activity for RhoA on the catalytic properties of PLC-delta1. Under in vitro conditions, ARHGAP6 protein activated PLC-delta1. ARHGAP6 protein bound PLC-delta1 and regulated its activity by masking the binding sites for inhibitory phospholipids. Moreover, ARHGAP6 increased the V(max) of PLC-delta1 and enhanced its response to Ca(2+) stimulation. A Western blot of immunoprecipitates from Cos-7 cells transfected with pcDNA3-ARHGAP6 and pcDNA3-PLCdelta1 showed the presence of ARHGAP6/PLC-delta1 complexes. The activity of PLC in cells overexpressing ARHGAP6 increased approximately 6-fold compared to control cells. The examination of ARHGAP6 expression in mononuclear cells isolated from the blood of patients with hypertension showed increased ARHGAP6 mRNA and protein levels compared to age-matched normotensive subjects. Enhanced expression of ARHGAP6 was associated with an elevated level of PLC activity and increased levels of IP(3) (1.6-fold) and DAG (2.3-fold). In summary, our data indicate that ARHGAP6 protein binds to and up regulates PLC-delta1 both under in vitro and in vivo conditions. Moreover, the elevated expression of ARHGAP6 provides possible explanation for the altered activity of PLC-delta1 in hypertension.

Topics: Adult; Animals; Blotting, Western; Chlorocebus aethiops; COS Cells; Diglycerides; Female; Gene Expression Regulation; GTPase-Activating Proteins; Humans; Hypertension; Inositol 1,4,5-Trisphosphate; Kinetics; Leukocytes, Mononuclear; Male; Phospholipase C delta; Phospholipids; Protein Binding; Recombinant Proteins; rhoA GTP-Binding Protein; RNA, Messenger

Aldosterone is a mineral corticoid hormone that is produced in response to angiotensin-II, and like angiotensin-II, stimulates inflammation, oxidative stress, and fibrosis by activating nuclear factor-kappaB and activating protein-1. Recent evidence, however, indicates that aldosterone stimulates phospholipase C and activates nuclear factor-kappaB and activating protein-1. Although the heme oxygenase system is cytoprotective, its effects on aldosterone-phospholipase C signaling in deoxycorticosterone acetate (DOCA-salt) hypertension, a model of aldosteronism, and spontaneously hypertensive rat, a genetic model of human essential hypertension, have not been fully characterized.. In the present study, the heme oxygenase inducer, hemin, was given to spontaneously hypertensive and deoxycorticosterone acetate hypertensive rats, and the effects on blood pressure, aldosterone, nuclear factor-kappaB, activating protein-1, phospholipase C, and inositol 1,4,5-triphosphate were examined.. Hemin therapy restored physiological blood pressure to spontaneously hypertensive rats (209.9 +/- 0.9 to 127.3 +/- 0.85 mmHg, n = 10, P < 0.01) and to deoxycorticosterone acetate salt hypertensive rats (195.7 +/- 1.8 vs.132.5 +/- 2.1 mmHg; P < 0.01, n = 10), but had no effect on age-matched normotensive Wistar-Kyoto or Sprague-Dawley strains. The antihypertensive effect was accompanied by enhanced heme oxygenase activity, upregulated cyclic guanosine monophosphate-protein kinase G signaling, increased superoxide dismutase activity, and the potentiation of total antioxidant capacity, whereas aldosterone, activating protein-1, and nuclear factor-kappaB were reduced. Furthermore, hemin suppressed phospholipase C activity, attenuated inositol 1,4,5-triphosphate, and reduced resting intracellular calcium in the aorta.. Collectively, our results suggest that the concomitant depletion of aldosterone, phospholipase C-inositol 1,4,5-triphosphate activity, resting intracellular calcium and the corresponding decline of inflammatory, and oxidative insults may account for the antihypertensive effects of hemin in deoxycorticosterone acetate hypertension and spontaneously hypertensive rats.

Topics: Aldosterone; Animals; Aorta; Blood Pressure; Calcium; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Down-Regulation; Heme Oxygenase (Decyclizing); Hemin; Hypertension; Inflammation; Inositol 1,4,5-Trisphosphate; Male; Oxidative Stress; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Receptor Cross-Talk; Second Messenger Systems; Type C Phospholipases; Up-Regulation

It is reported that dopamine promotes renal sodium excretion via activation of D1-like dopamine receptors located on the proximal tubules. In spontaneously hypertensive rats the natriuretic and diuretic response to exogenously administered and endogenously produced dopamine is reduced, which results from a diminished dopamine-induced inhibition of the enzyme, Na+,K+-ATPase. The present study was designed to examine dopamine-receptor mediated inhibition of Na+,K+-ATPase and its associated signal transduction pathway in the proximal tubules of Zucker obese and lean control rats. The obese animals were hypertensive, hyperinsulinaemic and hyperglycaemic compared with the lean rats. While dopamine caused inhibition of Na+,K+-ATPase activity in lean rats, this effect was significantly attenuated in the obese animals. There was significant reduction in D1-like receptor numbers in the basolateral membranes of obese rats compared with lean rats with no change in the affinity to the ligand [3H]SCH 23390 between the two groups of rats. Dopamine failed to stimulate G proteins as measured by [35S]GTPgammaS binding in the obese rats. Also, dopamine was unable to cause phospholipase-C activation in obese rats, but it did activate phospholipase-C in lean rats. These results show that reduction in D1-like receptor numbers and a defect in receptor-G protein coupling may account for the inability of dopamine to activate the D1-like receptor-coupled signal transduction pathway and cause inhibition of Na+,K+-ATPase in the obese hypertensive rats.

Topics: Animals; Benzazepines; Dopamine Antagonists; Guanosine 5'-O-(3-Thiotriphosphate); Hypertension; Inositol 1,4,5-Trisphosphate; Kidney; Male; Obesity; Rats; Rats, Zucker; Receptors, Dopamine D1; Reference Values; Signal Transduction; Sodium-Potassium-Exchanging ATPase

Inositol 1,4,5-trisphosphate receptor (IP(3)-receptor) is a calcium channel, transporting calcium from intracellular stores to the cytoplasm. In kidney, IP(3)-receptors are involved in the signal transduction of various hormones. In our work we studied the effect of immobilization stress on the IP(3)-receptor's protein content in renal cortex and the medulla of normotensive and hypertensive rats. We detected both mRNA and type 1 IP(3)-receptor protein in medulla, but not in renal cortex. We found that this receptor was approximately twice as abundant in normotensive as in genetically hypertensive rat kidney. Immobilization stress decreased the amount of type 1 IP(3)-receptor in the renal medulla of normotensive rats approximately five times, while no effect due to single and/or repeated stress was observed in the renal medulla of spontaneously hypertensive rats. The results indicate that expression of type 1 IP(3)-receptor in renal medulla is modulated by hypertension and immobilization stress.

Topics: Alternative Splicing; Animals; Blood Pressure; Body Weight; Calcium Channels; Down-Regulation; Hypertension; Immobilization; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kidney Cortex; Kidney Medulla; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Cytoplasmic and Nuclear; Stress, Physiological

The effects of hypoxanthine and xanthine oxidase-induced superoxide anion were evaluated on various signal transduction pathways in aortic smooth muscle cells (SMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Superoxide increased inositol 1,4,5-tris-phosphate (IP(3)) formation in a concentration- and time-dependent manner in both strains but more markedly in SMCs from SHR. Various antioxidants significantly decreased the superoxide-induced IP(3) formation in both strains. In addition, tyrosine kinase inhibitors, genistein and tyrphostin A25, inhibited the superoxide-induced IP(3) formation more markedly in SHR than in WKY. Moreover, superoxide decreased the basal level of cGMP to a greater extent in SHR and also suppressed the rise in cGMP induced by S-nitroso-N-acetylpenicillamine. In addition, the superoxide-induced increase in IP(3) formation was significantly inhibited by guanylyl cyclase stimulator S-nitroso-N-acetylpenicillamine but was potentiated by ODQ (a guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one) and KT5823 (a cGMP-dependent protein kinase inhibitor), with a greater effect in SHR. Finally, the superoxide-enhanced IP(3) formation was not accompanied by simultaneous changes in cAMP levels, and inhibition of the adenylyl cyclase pathway did not modify the superoxide-induced IP(3) formation. Our results thus demonstrate a stimulatory effect of superoxide on IP(3) formation, mediated by the tyrosine kinase-coupled phospholipase C(gamma) activity, and an inhibitory effect of superoxide on cGMP formation in vascular SMCs. The increased reactivity of the phospholipase C pathway and the decreased cross inhibition of the IP(3) pathway by cGMP in the presence of superoxide may underlie the altered functions of vascular SMCs in SHR.

Topics: Animals; Antioxidants; Aorta; Cells, Cultured; Cyclic AMP; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Hypoxanthine; Inositol 1,4,5-Trisphosphate; Muscle, Smooth, Vascular; Penicillamine; Protein-Tyrosine Kinases; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Signal Transduction; Superoxides; Xanthine Oxidase

We have previously demonstrated the vasorelaxant properties of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in normotensive and spontaneously hypertensive rat (SHR) aorta, although the mechanism(s) of action are not fully understood. Because endothelial dysfunction and increased intracellular free calcium concentration ([Ca2+]i) are seen in hypertensive rat aorta, we investigated the potential role of Ca2+ signaling, endothelium and derived factors, and the opening of potassium (K+) channels in EPA-induced relaxation. In the presence of extracellular Ca2+, EPA induced significant relaxations at >10 micromol/L (P<.01) in norepinephrine (NE) (10(-6) mol/L)-contracted aortic rings and at 30 micromol/L (P<.001) in high K+ (80 mmol/L)-contracted aortic rings. In the absence of extracellular Ca2+, EPA (10 to 30 micromol/L) inhibits the tonic component of NE-induced contraction (P<.0001). The relaxant properties of EPA in SHR aorta appear specific to Ca2+ release from an internal storage site associated with NE-induced tonic contraction. Further studies with the use of fura-2 to measure [Ca2+]i in cultured vascular smooth muscle (VSM) cells from SHR aorta indicated that EPA (30 micromol/ L)-pretreatment attenuated angiotensin II (50 nmol/ L)-induced Ca2+ transient by 95%, suggesting that an inhibitory effect on the Ca2+ signaling may underlie EPA-induced relaxation of the vessel preparation. In addition, EPA per se induced an increase in [Ca2+li with a duration of approximately 20 min in VSM cells, and the effect was not altered by removal of extracellular Ca2+. There was no increase in the level of inositol-1,4,5-trisphosphate in response to EPA (30 micromol/L). The actions of EPA are independent of endothelium-derived factors, cyclooxygenase metabolites, and activation of K+ channels since endothelium removal, N(omega)-nitro-L-arginine methyl ester hydrochloride, (L-NAME, 100 micromol/L), indomethacin (10 micromol/L), tetraethylammonium (1 mmol/L), and glibenclamide (10 micromol/L) did not affect EPA-induced vasodilation in NE-precontracted aortic rings. These results suggest that EPA directly modulates intracellular Ca2+ signaling in VSM cells, and that this may contribute to the vasorelaxant effect and, at least in part, the blood pressure-lowering effect of fish oil.

Topics: Angiotensin II; Animals; Aorta, Thoracic; Blood Pressure; Calcium; Cells, Cultured; Eicosapentaenoic Acid; Endothelium, Vascular; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Hypertension; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Male; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Norepinephrine; Potassium Channels; Rats; Rats, Inbred SHR; Vasoconstrictor Agents; Vasodilation

We investigated the mechanism of vasoconstrictor-induced endothelin-1 (ET-1) release from human umbilical vein endothelial cells (HUVECs) in serum from women with pregnancy-induced hypertension (PIH). We obtained serum samples from seven women with PIH, seven healthy nonpregnant women (NP), and seven normal pregnant women (NPIH). ET-1 and inositol 1,4,5-trisphosphate (IP3) were assayed by ET-1 ELISA and an IP3 3H assay system, respectively. ET-1 release from HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Angiotensin II (Ang II)- and epinephrine (Epi)-induced ET-1 release were significantly increased by PIH serum. IP3 production in HUVECs incubated with 10% serum (NP, NPIH, and PIH) was greater than that without human serum. Ang II- or Epi-induced IP3 production in HUVECs incubated with PIH serum was increased but not significantly compared to that with other sera. Our results suggest that increased ET-1 release from HUVECs incubated with human serum may be mediated by IP3 production, but that Ang II- or Epi-induced ET-1 release from HUVECs incubated with PIH serum may be mediated by another mechanism.

Topics: Endothelin-1; Endothelium, Vascular; Female; Humans; Hypertension; Inositol 1,4,5-Trisphosphate; Pregnancy; Pregnancy Complications, Cardiovascular

The effects of DHA treatment on intracellular Ca2+ dynamics in aortic smooth muscle cells isolated from young stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched normotensive Wistar Kyoto rats (WKY) were investigated. The resting intracellular Ca2+ concentration ([Ca2+]i) before stimulation and the peak [Ca2+]i induced by 5-HT, angiotensin II and depolarizing concentration of KC1 were higher in SHRSP than in WKY. When added to the culture medium for 2 days, DHA at a concentration of 30 microM significantly suppressed the peak [Ca2+]i induced by these stimulants in aortic smooth muscle cells isolated from WKY, whereas smooth muscle cells of SHRSP were refractory to the suppression. DHA had no suppressive effect on the 5-HT-induced increase in the inositol triphosphate production. The present study indicates that DHA can suppress receptor-mediated Ca2+ influx, at least, through the voltage-dependent channel, in vascular smooth muscle cells. Since the intracellular Ca2+ plays an important role in regulating vascular tone, the suppressive effect of DHA on [Ca2+]i in vascular smooth muscle cells may be contributed to the beneficial properties of DHA on cardiovascular disorders. The precise mechanisms of action remain to be elucidated.

Topics: Animals; Calcium; Cells, Cultured; Docosahexaenoic Acids; Hypertension; Inositol 1,4,5-Trisphosphate; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY

The changes of inositol phosphates in quiescent and GTP gamma S activated vascular smooth muscle cells (VSMC) derived from aorta of spontaneously hypertensive rat (SHR) were examined. The inositol phosphate assay revealed that there was no difference in the inositol-1, 4, 5-triphosphate (InsP3) level between the quiescent VSMCs of SHR and that of normotensive Wistar-Kyoto rats (WKY). G-protein activator GTP gamma S caused a dose- and time-dependent increase in the InsP3 level in VSMCs. Activated G proteins increased significantly the InsP3 level in VSMCs of SHR. In conclusion, the highly sensitive reactivity to GTP gamma S in the generation of InsP3 in VSMCs from SHR may be related to the possible abnomality of the number, substructure or function of some components in G proteins, phosphoinositide turnover.

Topics: Animals; Aorta, Thoracic; GTP-Binding Proteins; Hypertension; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Muscle, Smooth, Vascular; Rats; Rats, Inbred SHR; Rats, Inbred WKY

Recent studies have shown an enhanced signaling capacity of receptors coupled to pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G proteins) in immortalized B lymphoblasts from patients with essential hypertension. In the present study, we analyzed (1) whether such alterations would also be expressed in nontransformed cells of these individuals and (2) whether other G protein-mediated signaling pathways were also altered. Therefore, we established primary cultures of skin fibroblasts from previously characterized normotensive and hypertensive individuals (NT and HT cells, respectively). [Ca2+]i rises induced by lyso-phosphatidic acid (LPA), thrombin, and sphingosine-1-phosphate as well as the formation of inositol 1,4,5-trisphosphate and [3H]thymidine incorporation evoked by LPA were PTX sensitive and enhanced twofold in HT fibroblasts. In contrast, cellular responses induced by bradykinin, endothelin-1, and angiotensin II (all PTX insensitive) were similar in NT and HT cells. Formation of cAMP induced by stimulation of Gs with isoproterenol was identical in NT and HT cells. Western blot analysis yielded no evidence for an overexpression of G alpha i2, G alpha i3, G beta 2, and G beta 4. Furthermore, sequencing of cDNAs encoding for the ubiquitously expressed PTX-sensitive G protein subunits G alpha i2, G alpha i3, G beta 1, and G beta 2 from NT and HT cell lines yielded no evidence for mutations in these genes. Although the molecular mechanisms remain to be defined, these data support the concept of a selective enhancement of signal transduction via PTX-sensitive G proteins in essential hypertension.

Topics: Base Sequence; Calcium; Cells, Cultured; Cloning, Molecular; Cyclic AMP; DNA; Fibroblasts; GTP-Binding Proteins; Humans; Hypertension; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Mutation; Osmolar Concentration; Receptors, Cell Surface; Reference Values; Signal Transduction; Skin

Because the phosphatidylinositol pathway may be part of the signaling system associated with stretch-induced release of atrial natriuretic peptide (ANP), we tested the hypothesis that formation of the intermediate inositol-1,4,5-trisphosphate (IP3) is impaired when ANP release is decreased in response to atrial stretch in hearts from aging genetically hypertensive (GH) rats. Immunoreactive ANP release into the coronary effluent and IP3 levels were studied in cardiac tissues of isolated perfused hearts from normotensive control (WAG) or GH rats aged 4, 11, or 16 months. Left atria were repeatedly distended and released with a latex balloon. ANP was measured in coronary effluent, and IP3 was measured in cardiac tissues. In all age groups, stretch and relief of stretch evoked considerably less ANP release in spontaneously beating hearts from GH than from WAG rats. Hearts from GH rats aged 16 months released no ANP, but electrical pacing restored some stretch-induced ANP secretion. With repeated stretch and release of stretch of the left atrium for 2 min, IP3 levels increased in left atrial tissue in WAG but not in GH hearts of all age groups. IP3 levels in (unstretched) left ventricles were much lower than in left atria and were unaltered by atrial stretch. In aging GH rats, the capacity to release ANP on atrial stretch is largely lost, in association with complete suppression of stimulus-induced increase in IP3 levels. These data support a role for IP3 in stretch-mediated atrial ANP secretion and suggest a progressive uncoupling of this signaling pathway in aging hypertensive rats.

Topics: Aging; Analysis of Variance; Angioplasty, Balloon; Animals; Atrial Function; Atrial Natriuretic Factor; Blood Pressure; Body Weight; Cardiac Pacing, Artificial; Compliance; Disease Models, Animal; Hypertension; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Myocardium; Organ Size; Rats; Rats, Wistar

Endothelin (ET) B-type receptor-mediated signal transduction after stimulation with ET-3 was examined in cultured aortic endothelial cells obtained from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. The purpose of this study was to elucidate ETB receptor-mediated response in endothelial cells from hypertensive rat models. Non-isopeptide-selective displacement and affinity in these binding experiments suggest that aortic endothelial cell receptors for ET-3 correspond to ETB receptor subtypes. These receptors for ET-3 were similar in WKY and SHR endothelial cells. ETB receptor mRNA expression in cultured endothelial cells was also similar in WKY and SHR. However, the cytosolic free Ca2+ level in the absence of extracellular Ca2+ as well as the inositol 1,4,5-trisphosphate level in response to ET-3 were greater in endothelial cells from SHR than in those from WKY. Phospholipase C and protein kinase C activities after stimulation with ET-3 were also greater in SHR than in WKY. The 6-ketoprostaglandin F1 alpha production was also augmented in SHR, although nitric oxide formation and guanosine 3',5'-cyclic monophosphate production after stimulation with ET-3 were similar in WKY and SHR. We conclude that the phosphoinositide turnover signaling stimulated by ET-3 is augmented in cultured aortic endothelial cells from SHR compared with those from WKY.

Topics: Animals; Aorta; Calcium; Cells, Cultured; Endothelins; Endothelium, Vascular; Epoprostenol; Hypertension; Inositol 1,4,5-Trisphosphate; Osmolar Concentration; Peptides; Phosphatidylinositols; Phosphorylation; Protein Kinase C; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Signal Transduction; Type C Phospholipases

The vasoconstrictor responses to alpha-1 adrenoceptor agonists, binding behavior of alpha-1 adrenoceptors and postreceptor events in the mesenteric vasculature from deoxycorticosterone acetate-salt hypertensive rats were studied. The reactivity of perfused mesenteric artery to norepinephrine (NE) and phenylephrine, but not KCl, was enhanced significantly in the hypertensive rats compared with control rats. Prazosin antagonized the pressor response to NE more effectively in the hypertension than in the control. [3H]Prazosin binding was saturable and a single class of specific sites. Scatchard analysis revealed that the dissociation constant for [3H]prazosin was lower and the maximum binding capacity was greater in the hypertensive rats than in the control rats. The NE-stimulated phosphatidylinositol hydrolysis, estimated by measuring inositol 1,4,5-triphosphate (IP3) accumulation, was greater in the hypertensive rat artery compared with the control one. The IP3-induced contraction in the beta-escin-treated mesenteric large resistance vessel was smaller in the hypertensive rats. The vasoconstrictor response to phorbol 12,13-dibutyrate of the perfused mesenteric artery was larger in the hypertensive animals than in the control. Staurosporine antagonized the phorbol 12,13-dibutyrate-induced vasoconstriction in preparations from both rats. These results suggest that the increases in the number and affinity of alpha-1 adrenoceptors may result in an enhanced phosphatidylinositol hydrolysis accounting for an increased vascular reactivity to alpha-1 adrenoceptor agonists in deoxycorticosterone acetate-salt hypertensive rats. Furthermore, the enhancement of vasoconstrictor mechanism mediated by protein kinase C pathway may also contribute to vascular hyper-reactivity to NE, whereas the decreased IP3-induced contraction may function to minimize the hyper-reactivity observed in this model of experimental hypertension.

Topics: Animals; Calcium; Desoxycorticosterone; Hypertension; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Mesenteric Arteries; Norepinephrine; Phorbol 12,13-Dibutyrate; Prazosin; Protein Kinase C; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Sodium Chloride; Vasoconstriction

High intracellular levels of free calcium (Ca2+) are found in cells of hypertensive patients, and these levels have been correlated with increased blood pressure. In the present study, we characterized inositol 1,4,5-trisphosphate (InsP3) receptors in aorta and heart microsomes of Long-Evans rats. We also studied this receptor in some organs of spontaneously hypertensive rats (SHR) to clarify the role this important component of the mechanism of Ca2+ regulation has in hypertension. Microsomal fractions were prepared from aorta, heart, adrenal cortex (organs directly involved in the regulation of blood pressure), and cerebellum (control organ, not directly involved) of 14-week-old SHR and Wistar-Kyoto (WKY) rats. InsP3, receptors were studied in each microsomal fraction by a radioligand binding method with [3H]InsP3. The properties of InsP3 binding sites in each tissue were consistent with those of well-characterized InsP3 receptors: 1) InsP3 binding was specific and saturable, 2) InsP3 binding increased upon pH elevation, 3) InsP3 binding was inhibited by heparin, and 4) InsP3 had a higher binding affinity than InsP4. No significant difference of binding affinity or of maximal binding capacity between WKY and SHR was observed in microsomes from heart, adrenal cortex, and cerebellum. It is interesting that InsP3 binding capacity of SHR aorta microsomes was significantly higher (1.6 fold) than that of WKY aorta microsomes. These results suggest that increased InsP3 receptor activity in vascular smooth muscle of SHR may contribute to the elevation of blood pressure. Further studies are needed to assess the significance of this observation as regards the mobilization of intracellular Ca2+.

Topics: Animals; Aorta; Hypertension; Inositol 1,4,5-Trisphosphate; Male; Membranes; Microsomes; Muscle, Smooth, Vascular; Myocardium; Rats; Rats, Inbred SHR; Rats, Inbred WKY

The accumulations of inositol-1,4,5-trisphosphate (IP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) after hormonal stimulation may have a physiological role, possibly by alteration of Ca2+ levels in cardiac tissue. But the accumulation of inositol polyphosphate in a pathophysiological condition has not been studied. We investigated phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism in hypertrophic cardiac myocytes, and clarified that the accumulations of IP3, IP4 and diacylglyceride after stimulation with norepinephrine were significantly enhanced in isolated myocytes from spontaneously hypertensive rat heart. Phospholipase C activity increased with age in SHRSP heart cells. These data suggest that PI turnover pathways, which can be mediated by both phosphatidylinositol-4,5-bisphosphate and diacylglyceride, may play an important role in development of hypertrophy in the hearts of rats with spontaneous hypertension.

Topics: Animals; Cardiomegaly; Hypertension; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Myocardium; Norepinephrine; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Type C Phospholipases

Previous studies have shown that chronic subcutaneous administration of heparin significantly reduces blood pressure in hypertensive rats. The intracellular mechanisms of how heparin prevents smooth muscle cell proliferation remain unclear. This study was designed to examine whether heparin affects endothelin-1 action and production, to further elucidate the mechanism of the antihypertensive effect of heparin. Four-week treatment with heparin (300 U/day sc) significantly decreased blood pressure in spontaneously hypertensive rats (SHR; 199 +/- 8 vs. 164 +/- 9 mmHg; P < 0.001) and completely blunted pressor response to endothelin-1 in SHR. Heparin treatment did not decrease blood pressure response nor did it attenuate blood pressure responses to endothelin-1 in Wistar-Kyoto rats (WKY). Heparin significantly suppressed endothelin-1-induced increase in intracellular calcium concentration and inositol 1,4,5-trisphosphate level in cultured vascular smooth muscle cells in a dose-dependent manner and endothelin-1 release from cultured endothelial cells. These inhibitions were significantly more pronounced in SHR than in WKY. In conclusion, our findings indicate that the antihypertensive effect of heparin is mediated, at least in part, by the inhibition of endothelin-1 action on vascular smooth muscle cells and endothelin-1 production from endothelial cells.

Topics: Animals; Aorta; Binding Sites; Blood Pressure; Calcium; Cells, Cultured; Endothelins; Endothelium, Vascular; Heart Rate; Heparin; Hypertension; Inositol 1,4,5-Trisphosphate; Male; Muscle, Smooth, Vascular; Osmolar Concentration; Radioimmunoassay; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Time Factors