Elevation of intravascular pressure causes depolarization and constriction (myogenic tone) of small arteries and arterioles, and this response is a key element in blood flow regulation. However, the nature of pressure-induced depolarization has remained elusive. In the present study, we provide evidence that a transient receptor potential channel (TRPC6) homologue has a major role in this depolarizing response to pressure. Antisense oligodeoxynucleotides to TRPC6 decreased TRPC6 protein expression and greatly attenuated arterial smooth muscle depolarization and constriction caused by elevated pressure in intact cerebral arteries. Suppressing the expression of this channel protein also reduced the current density of a major cation current in resistance artery smooth muscle cells. We propose that TRPC6 channels play an essential role in regulation of myogenic tone. O riginally described by Bayliss 100 years ago, 1 myogenic tone develops when intravascular pressure is increased. Myogenic tone plays a key role in regulation of tissue blood flow in vivo 2 and thus is of major significance in cardiovascular control. Elevation of intravascular pressure depolarizes the smooth muscle cells within the arterial wall. 3 The depolarization activates dihydropyridine-sensitive (L-type) voltage-dependent Ca 2ϩ channels, 4 which leads to increased intracellular Ca 2ϩ and vasoconstriction. Although depolarization is key to myogenic tone, the molecular identity of the ion channels involved in this response is an important unresolved issue in vascular biology. However, the recently identified mammalian transient receptor potential channels (TRPCs) 5 are good candidates for this role in arterial smooth muscle. Members of this family of cation channels are found in many different tissue types including vascular cells. 6 -9 The TRPC6 homologue is highly expressed in vascular smooth muscle, and these channels share many of the biophysical properties of vascular cation currents. 6,7,10,11 In the present study, we have tested the hypothesis that TRPC6 channels are involved in the pressureinduced depolarization that increases myogenic tone. Materials and MethodsCerebellar and posterior cerebral arteries (150 m, inner diameter) from 12-to 16-week-old Sprague-Dawley rats (Charles River Laboratories, St Constant, Canada) were studied. All animal use procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee at the University of Vermont. For RT-PCR analysis, total RNA was extracted from arterial segments (Ϸ3 mm long) or Ϸ100 enzymatically isolated myocytes, and first-strand cDNA was synthesized. Forward and reverse primers specific for TRPC6 were TRPC6F 5Ј-GTGCCAAGTCCA-AAGTCCCTGC-3Ј and TRPC6R 5Ј-CTGGGCCTGCAGTA-CGTATC-3Ј. These primers yield a 315-bp TRPC6 cDNA product.To assess TRPC protein expression, arteries were homogenized. Solubilized proteins were then separated by electrophoresis and detected using polyclonal antibodies to TRPC3 (anti-rabbit, 1:1000 dilution, Alomone La...

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The human tumor necrosis factor alpha (TNF-␣) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-␣ enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-␣ gene transcription and selectively associates with the TNF-␣ promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-␣ transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases.