In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/ signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II-or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors.Previous work by our laboratory (1-6) on cultured rat aortic vascular smooth muscle cells (VSMC) 1 and phenotypically similar glomerular mesangial cells has shown that protein tyrosine phosphorylation plays a critical role in angiotensin II (Ang II)-mediated intracellular signaling cascades. This is true despite the fact that G protein-coupled receptors in general and the Ang II AT 1 receptor in particular possess no intrinsic tyrosine kinase activity. It is also now recognized that Ang II can act not only as a vasoactive peptide but also as a growth factor. In particular, Ang II has been shown to stimulate proliferative and hypertrophic growth in VSMC, glomerular mesangial cells, cardiac fibroblasts, and myocytes via AT 1 receptor binding (4, 7-9). Like classic growth factors (e.g. platelet-derived growth factor (PDGF) and epidermal growth factor) and some cytokines (e.g. interferons and interleukins) (4, 8 -10), Ang II is also capable of stimulating a rapid increase in the mRNA levels of c-fos, an early growth response gene implicated in VSMC proliferation (4,7,8). However, the Ang II-stimulated intracellular signaling cascades responsible for c-fos induction and therefore proliferation in VSMC have not been well defined.One candidate mitogenic signaling cascade involves the activation of the small GTP-binding protein, Ras, which is traditionally mediated via classic growth factor receptors (4). Ras activation promotes the formation of a membrane-bound complex with Raf-1 (a serine/threonine protein kinase). Subsequent tyrosine phosphorylation of Raf-1 leads to its activation and the sequential stimulati...

1) modulation of apical Na+ permeability by luminal Na+ does not require direct interaction of Na+ with the channel protein but, rather, appears to involve an intracellular regulatory pathway, 2) relieving self-inhibition alters both the number and kinetics of single Na+ channels, 3) the effect of low Na+ must be modulated via decreased apical Na+ entry and intracellular Na+, since amiloride yielded similar results, 4) changes in intracellular Na+ probably affect Na+ channel activity via cytosolic Ca2+, 5) the effects of decreasing luminal Na+ are reversed by PKC activators and mimicked by PKC inhibitors suggesting a possible role for PKC in Na+ self-inhibition.

An early event in signaling by the G-protein-coupled angiotensin II (Ang II) AT 1 receptor in vascular smooth muscle cells is the tyrosine phosphorylation and activation of phospholipase C␥1 (PLC␥1). In the present study, we show that stimulation of this event by Ang II in vascular smooth muscle cells is accompanied by binding of PLC␥1 to the AT 1 receptor in an Ang II-and tyrosine phophorylation-dependent manner. The PLC␥1-AT 1 receptor interaction appears to depend on phosphorylation of tyrosine 319 in a YIPP motif in the C-terminal intracellular domain of the AT 1 receptor and binding of the phosphorylated receptor by the most C-terminal of two Src homology 2 domains in PLC␥1. PLC␥1 thus binds to the same site in the receptor previously identified for binding by the SHP-2 phosphotyrosine phosphatase⅐JAK2 tyrosine kinase complex. A single site in the C-terminal tail of the AT 1 receptor can, therefore, be bound in a ligand-dependent manner by two different downstream effector proteins. These data demonstrate that G-protein-coupled receptors can physically associate with intracellular proteins other than G proteins, creating membrane-delimited signal transduction complexes similar to those observed for classic growth factor receptors.Growth factor receptors belong to a family of receptors that contain an extracellular ligand binding domain, a single transmembrane portion, and a large intracellular tyrosine kinase catalytic domain. Ligand-induced receptor autophosphorylation promotes the interaction of the intracellular domains of the receptors with a number of downstream effector proteins or enzymes. Typically, these proteins contain one or more domains known as Src homology 2 (SH2) 1 domains. Among these SH2 domain-containing proteins are phosphoinositide-specific phospholipase C␥ (PLC␥), the 85-kDa subunit of phosphatidylinositol 3-kinase, GTPase-activating proteins, growth factor receptor binding protein 2, the phosphotyrosine phosphatase SHP-2, and members of the nonreceptor Src family of tyrosine kinases (1, 2). Autophosphorylation of growth factor receptors occurs on defined tyrosine residues. These phosphorylated residues function to initiate cellular signaling cascades by acting as high affinity binding sites for the SH2 domains of various effector proteins. The selectivity of the receptor-effector interaction is determined, not only by the phosphorylated tyrosine residue in the receptor but also by the three amino acids Cterminal to the phosphorylated tyrosine and by the structure of the SH2 domain of the interacting protein. For example, one of the identified sites for binding of the SH2 domains of PLC␥1 to the platelet-derived growth factor ␣ and ␤ receptors is a YIPP motif present in the receptors at residues 1018 -1021 and 1021-1024, respectively. Phosphorylation of tyrosines 1018 and 1021 in these motifs promotes binding of PLC␥1 to the platelet-derived growth factor receptor and tyrosine phosphorylation and activation of the enzyme (3, 4).Another family of cell surface receptors are the G-protein...

We used patch-clamp methods to investigate the effects of basolateral endothelin-1 (ET-1) on the amiloride-sensitive Na+ channel in A6 distal nephron cells. One hundred picomolar ET-1 decreased channel activity via an increase in mean time closed (P < 0.01, n = 10). Channel inhibition by pM ET-1 was mimicked by an ET-B receptor agonist (P < 0.05, n = 7) and was prevented by ET-B antagonists (P = 0.14, n = 10) but not by an ET-A antagonist (P < 0.05, n = 4). With the inhibitory ET-B receptor blocked, higher doses of ET-1 (10 nM) actually increased channel activity through an increase in mean time open (P < 0.001, n = 12). The current-voltage relationship and the number of channels were not changed by basolateral ET-1 exposure. We conclude that 1) basolateral ET-1 regulates amiloride-sensitive Na+ channels; 2) binding of picomolar ET-1 to ET-B receptors inhibits, whereas the binding of nanomolar ET-1 to a different ET receptor (likely ET-A) stimulates, channel activity; and 3) these dose-dependent, distal nephron responses provide a potential mechanism for the in vivo natriuresis and antinatriuresis observed in response to "subpressor" and "pressor" concentrations of ET-1, respectively.

The binding of vasoactive peptides to their respective G protein-coupled receptors has been implicated in the pathogenesis of vascular smooth muscle cell proliferation, leading to the development of hypertension, arteriosclerosis, and restenosis after vascular injury. We previously showed that the cytosolic tyrosine kinase pp60c-src is crucial for angiotensin II (ANG II)-induced activation of the protooncogene p21ras. Therefore, we investigated the role of pp60c-src and p21ras in rat aortic smooth muscle cell proliferation induced by several G protein-coupled receptors. ANG II, endothelin-1, or thrombin increased cell proliferation and DNA synthesis. Electroporation of anti-pp60c-src antibodies into cells abolished proliferation in response to these G protein-coupled receptor ligands but not in response to platelet-derived growth factor-BB (PDGF-BB). In contrast, electroporation of anti-p21ras antibody completely blocked DNA synthesis and cell proliferation in response to ANG II, endothelin-1, thrombin, and PDGF-BB. Our data indicate that the pp60c-src tyrosine kinase is necessary and specific for vascular smooth muscle cell proliferation and DNA synthesis in response to G protein-coupled receptors but not classic growth factor receptors.