Na؉ /H ؉ exchanger isoform-1 (NHE1), the ubiquitous form of the Na ؉ /H ؉ exchanger, has increased activity in hypertensive patients and in animal models of hypertension. Furthermore, NHE1 is activated in cells stimulated with growth factors. We showed previously that activation of the exchanger is dependent on phosphorylation of serine 703 (Ser(P) 703 ) by p90 ribosomal S6 kinase (RSK). Because the NHE1 sequence at Ser(P) 703(RIGSDP) is similar to a consensus sequence (RSXSXP) specific for 14-3-3 ligands, we evaluated whether serum stimulated 14-3-3 binding to NHE1. Five different GST-NHE1 fusion proteins spanning amino acids 515-815 were phosphorylated by RSK and used as ligands in a far Western analysis; only those containing Ser(P) 703 exhibited high affinity 14-3-3 binding. In PS127A cells (NHE1-overexpressing Chinese hamster fibroblasts) stimulated with 20% serum, NHE1 co-precipitation with GST-14-3-3 fusion protein increased at 5 min (5.2 ؎ 0.4-fold versus control; p < 0.01) and persisted at 40 min (3.9 ؎ 0.3-fold; p < 0.01). We confirmed that binding occurs at the RIGSDP motif using PS120 (NHE1 null) cells transfected with S703A-NHE1 or P705A-NHE1 (based on data indicating that 14-3-3 binding requires phosphoserine and ؉2 proline). Serum failed to stimulate association of 14-3-3 with these mutants. A GST-NHE1 fusion protein was phosphorylated by RSK and used as a ligand to assess the effect of 14-3-3 on protein phosphatase 1-mediated dephosphorylation of Ser(P) 703 . GST-14-3-3 limited dephosphorylation (66% of initial state at 60 min) compared with GST alone (27% of initial state; p < 0.01). The protective effect of GST-14-3-3 was lost in the GST-NHE1 P705A mutant. Finally, the baseline rate of pH recovery in acid-loaded cells was equal in unstimulated cells expressing wild-type or P705A-NHE1. However, activation of NHE1 by serum was dramatically inhibited in cells expressing P705A-NHE1 compared with wild-type (0.13 ؎ 0.02 versus 0.48 ؎ 0.06 mmol of H ؉ /min/liter, p < 0.01). These data suggest that 14-3-3 binding to NHE1 participates in serum-stimulated exchanger activation, a new function for 14-3-3. NHE11 is the ubiquitous form of the Na ϩ /H ϩ exchanger, responsible for regulation of intracellular pH and cell volume. NHE1 has increased activity in hypertensive patients and in animal models of hypertension, and the exchanger can be stimulated by various exogenous factors. In several examples of increased NHE1 activity, the primary change is not in NHE1 expression but in post-translational regulation of activity. In immortalized cells from hypertensive patients, enhanced NHE1 activity appears to reflect increased V max rather than increased NHE1 mRNA (1). Similarly, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) show an increased V max compared with their normotensive Wistar-Kyoto (WKY) counterparts but do not overexpress NHE1 mRNA or protein (2-4). Furthermore, there is no evidence for genetic linkage between the Na ϩ /H ϩ exchanger gene and human hypertension (5), a...

We have tested the hypothesis that growth factor signaling pathways are augmented in hypertension, a disease associated with vascular smooth muscle cell growth. Thoracic aorta was dissected from deoxycorticosterone acetate-salt (DOCA-salt) and one kidney, one clip (1K, 1C) hypertensive rats and from sham normotensive rats for use in isolated tissue bath experiments. Systolic blood pressure was significantly higher in DOCA-salt and 1K, 1C than in normotensive sham rats: 192 ± 7, 185 ± 10, and 117 ± 4 mmHg, respectively. Although virtually no contraction to epidermal growth factor (EGF) was observed in endothelium-denuded sham rat aorta [1 ± 1% phenylephrine (PE) (10 μmol/l)-induced contraction], the maximal EGF-induced contraction was 45 ± 7% in endothelium-denuded aorta from DOCA-salt hypertensive rats and 39 ± 7% in aorta from 1K, 1C rats. Although slightly attenuated, a contraction to EGF was still observed in endothelium-intact aortic strips from 28-day DOCA-salt hypertensive rats. We also conducted concentration-response curves to EGF on days 1, 3, 5, 7, 14, and 21 of DOCA-salt therapy. A significant contraction to EGF in aorta from DOCA-salt rats was observed on day 14, when DOCA-salt rats had significantly higher blood pressure than sham rats: 188 ± 6 and 122 ± 3 mmHg, respectively. Transforming growth factor-α, an agonist of the EGF receptor, contracted DOCA-salt rat aorta (30 ± 7% PE-induced contraction) but not sham aorta (3 ± 3%). The EGF receptor tyrosine kinase inhibitor 4,5-dianilinophthalimide (10 μmol/l), the mitogen-activated protein kinase kinase inhibitor PD-098059 (10 μmol/l), and the L-type voltage-gated calcium channel inhibitor diltiazem (1 mol/l), but not the cyclooxygenase inhibitor indomethacin (10 μmol/l), virtually abolished EGF-induced contraction (85, 98, and 99% reduction, respectively). These data support a striking difference in EGF signaling between normotensive and hypertensive animals. Furthermore, they provide evidence that growth factors should be considered vasoconstrictors as well as growth modulators in hypertension.

The present studies test the hypothesis that contraction to EGF is dependent on mineralocorticoids and/or an elevation in systolic blood pressure (SBP). Endothelium-denuded thoracic aortas from sham normotensive, N(omega)-nitro-L-arginine (L-NNA) hypertensive, Wistar-Kyoto (WKY), and spontaneously hypertensive rats (SHR) were used in isolated tissue-bath experiments. Maximal contraction to epidermal growth factor [EGF; percentage of phenylephrine (PE; 10 umol/l)-induced contraction] was greater in strips from L-NNA (32 +/- 5%) and SHR (53 +/- 8%) rats compared with sham and WKY rats (17 +/- 1 and 12 +/- 4%, respectively). Wistar-Furth rats became only mildly hypertensive when given DOCA salt (134 +/- 6 mmHg) compared with Wistar rats (176 +/- 9 mmHg), but aortas from both strains had a similarly enhanced contraction to EGF (approximately 9 times the maximal contraction of sham aorta). Furthermore, in vitro incubation of aortas from Wistar and Wistar-Furth rats with aldosterone (10 nmol/l) increased EGF-receptor mRNA expression by >50%. These data indicate that arterial contraction to EGF may occur independent of hypertension and be stimulated by mineralocorticoids.

Abstract-Epidermal growth factor (EGF) causes contraction in arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats but not in normotensive sham rats. We hypothesized that an increase in the number of EGF receptors (EGFRs) in arteries from DOCA-salt rats enables the observed contraction to EGF to occur. DOCA-salt rats had a systolic blood pressure Ͼ170 mm Hg, whereas all sham rats had a systolic blood pressure Ͻ125 mm Hg. Thoracic aorta were removed for measurement of isometric force, EGFR mRNA levels, and EGFR protein levels. EGF caused a significant contraction in endothelium-denuded aorta from DOCA-salt rats (38Ϯ7% of maximal phenylephrine-induced [10 mol/L] contraction) compared with aorta from sham rats (4Ϯ2%). The EGFR tyrosine kinase-specific inhibitors 4,5-dianilinophthalimide (10 mol/L) and AG1478 (250 nmol/L) reduced contraction in aorta from DOCA-salt by 85Ϯ14% and 65Ϯ10%, respectively. EGFR mRNA in DOCA-salt aorta was increased 4.2-fold compared with that in sham aorta. However, Western analyses of membrane-enriched and whole-tissue lysate of aorta from sham and DOCA-salt revealed no statistical difference in the density of EGFR protein between sham and DOCA-salt aorta. These data refute our hypothesis and suggest that a change downstream of EGFR is responsible for enabling EGF-induced contraction in hypertension. Key Words: EGF Ⅲ mineralocorticoid Ⅲ artery Ⅲ tyrosine kinase E pidermal growth factor (EGF) is a 6-kDa protein derived from a larger propeptide found in platelets, kidneys, and salivary glands. EGF binds 1 of 4 recognized receptorsErbB1 (EGF receptor [EGFR], Her-1), the orphan receptor ErbB2 (Neu, Her-2), ErbB3 (Her-3), and ErbB4 (Her-4)-to cause receptor dimerization and intermolecular autophosphorylation through activation of the cytoplasmic tyrosine kinase integral to these receptors. 1,2 The autophosphorylated dimer recruits adaptor proteins to lead to activation of multiple signaling pathways, including phosphoinositide 3-kinase, Janus kinase/signal transducer, and activator of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) cascades. 3 Dimerization is necessary for receptor activation and thus physical clustering of receptors through increased expression or reorganization could potentially facilitate activation of these receptors.Growth factors such as EGF are vascular smooth muscle cell mitogens. 4 Recently, growth factors have been discovered to modulate smooth muscle contractility. 5-7 Inappropriate vascular growth or remodeling and enhanced contractility are commonly observed in forms of human and experimental hypertension. 8 We previously presented the novel finding of a significant contraction to EGF in arteries from hypertensive but not normotensive rats 9 and herein test the hypothesis that contraction to EGF is enabled in arteries of hypertensive animals because of an increased expression of the primary receptor for EGF, EGFR. It is becoming increasingly evident that the EGFR serves as a relay station for signaling of crucial vasoactive ...