Abstract-Changes in membrane tension resulting from membrane stretch represent one of the key elements in blood flow regulation in vascular smooth muscle. However, the molecular mechanisms involved in the regulation of membrane stretch remain unclear. In this study, we provide evidence that a vanilloid receptor (TRPV) homologue, TRPV2 is expressed in vascular smooth muscle cells, and demonstrate that it can be activated by membrane stretch. Cell swelling caused by hypotonic solutions activated a nonselective cation channel current (NSCC) and elevated intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in freshly isolated cells from mouse aorta. Both of these signals were blocked by ruthenium red, an effective blocker of TRPVs. The absence of external Ca 2ϩ abolished this increase in [Ca 2ϩ ] i caused by the hypotonic stimulation and reduced the activation of NSCC. Significant immunoreactivity to mouse TRPV2 protein was detected in single mouse aortic myocytes. Moreover, the expression of TRPV2 was found in mesenteric and basilar arterial myocytes. Treatment of mouse aorta with TRPV2 antisense oligonucleotides resulted in suppression of hypotonic stimulationinduced activation of NSCC and elevation of [Ca 2ϩ ] i as well as marked inhibition of TRPV2 protein expression. In Chinese hamster ovary K1 (CHO) cells transfected with TRPV2 cDNA (TRPV2-CHO), application of membrane stretch through the recording pipette and hypotonic stimulation consistently activated single NSCC. Moreover, stretch of TRPV2-CHO cells cultured on an elastic silicon membrane significantly elevated [Ca 2ϩ ] i . These results provide a strong basis for our purpose that endogenous TRPV2 in mouse vascular myocytes functions as a novel and important stretch sensor in vascular smooth muscles. Key Words: TRPV2 Ⅲ vanilloid receptor Ⅲ mouse aorta Ⅲ membrane stretch Ⅲ vascular smooth muscle D etection of mechanical stimuli is essential for diverse biological functions including audition, touch, and maintenance of vascular myogenic tone. In the latter, elevation of intravascular pressure depolarizes vascular smooth muscle cells via membrane stretch. 1,2 This depolarization activates voltage-dependent L-type Ca 2ϩ channels (VDCC) and increases [Ca 2ϩ ] i , resulting in vasoconstriction and/or myogenic tone. 3 A large component of the elevation of [Ca 2ϩ ] i by myogenic tone can be inhibited by blockers of VDCC. However, some components are resistant to these agents, and instead can be accounted for by a separate nonselective cation channel that is permeable to Ca 2ϩ and also is activated by the intravascular pressure. 4,5 Because stretchactivated channels play obligatory roles in regulation of the myogenic tone, extensive studies have been performed to identify the molecular entity of these channels. Originally, a yeast MID1 gene product (MID1) was shown to be a eukaryotic stretch-activated channel, because CHO cells expressing MID1 responded to membrane stretch. 6 A member of the transient receptor potential channels (TRP), specifically TRPC6, is sensitive ...

Background and purpose: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta.

Despite the important roles played by ventricular fibroblasts and myofibroblasts in the formation and maintenance of the extracellular matrix, neither the ionic basis for membrane potential nor the effect of modulating membrane potential on function has been analyzed in detail. In this study, whole cell patch-clamp experiments were done using ventricular fibroblasts and myofibroblasts. Time- and voltage-dependent outward K(+) currents were recorded at depolarized potentials, and an inwardly rectifying K(+) (Kir) current was recorded near the resting membrane potential (RMP) and at more hyperpolarized potentials. The apparent reversal potential of Kir currents shifted to more positive potentials as the external K(+) concentration ([K(+)](o)) was raised, and this Kir current was blocked by 100-300 muM Ba(2+). RT-PCR measurements showed that mRNA for Kir2.1 was expressed. Accordingly, we conclude that Kir current is a primary determinant of RMP in both fibroblasts and myofibroblasts. Changes in [K(+)](o) influenced fibroblast membrane potential as well as proliferation and contractile functions. Recordings made with a voltage-sensitive dye, DiBAC(3)(4), showed that 1.5 mM [K(+)](o) resulted in a hyperpolarization, whereas 20 mM [K(+)](o) produced a depolarization. Low [K(+)](o) (1.5 mM) enhanced myofibroblast number relative to control (5.4 mM [K(+)](o)). In contrast, 20 mM [K(+)](o) resulted in a significant reduction in myofibroblast number. In separate assays, 20 mM [K(+)](o) significantly enhanced contraction of collagen I gels seeded with myofibroblasts compared with control mechanical activity in 5.4 mM [K(+)](o). In combination, these results show that ventricular fibroblasts and myofibroblasts express a variety of K(+) channel alpha-subunits and demonstrate that Kir current can modulate RMP and alter essential physiological functions.

KCNQ gene expression was previously shown in various rodent blood vessels, where the products of KCNQ4 and KCNQ5, Kv7.4 and Kv7.5 potassium channel subunits, respectively, have an influence on vascular reactivity. The aim of this study was to determine if small cerebral resistance arteries of the rat express KCNQ genes and whether Kv7 channels participate in the regulation of myogenic control of diameter. Quantitative reverse transcription polymerase chain reaction (QPCR) was undertaken using RNA isolated from rat middle cerebral arteries (RMCAs) and immunocytochemistry was performed using Kv7 subunit-specific antibodies and freshly isolated RMCA myocytes. KCNQ4 message was more abundant than KCNQ5 = KCNQ1, but KCNQ2 and KCNQ3 message levels were negligible. Kv7.1, Kv7.4 and Kv7.5 immunoreactivity was present at the sarcolemma of freshly isolated RMCA myocytes. Linopirdine (1 μm) partially depressed, whereas the Kv7 activator S-1 (3 and/or 20 μm) enhanced whole-cell Kv7.4 (in HEK 293 cells), as well as native RMCA myocyte Kv current amplitude. The effects of S-1 were voltage-dependent, with progressive loss of stimulation at potentials of >−15 mV. At the concentrations employed linopirdine and S-1 did not alter currents due to recombinant Kv1.2/Kv1.5 or Kv2.1/Kv9.3 channels (in HEK 293 cells) that are also expressed by RMCA myocytes. In contrast, another widely used Kv7 blocker, XE991 (10 μm), significantly attenuated native Kv current and also reduced Kv1.2/Kv1.5 and Kv2.1/Kv9.3 currents. Pressurized arterial myography was performed using RMCAs exposed to intravascular pressures of 10-100 mmHg. Linopirdine (1 μm) enhanced the myogenic response at ≥20 mmHg, whereas the activation of Kv7 channels with S-1 (20 μm) inhibited myogenic constriction at >20 mmHg and reversed the increased myogenic response produced by suppression of Kv2-containing channels with 30 nm stromatoxin (ScTx1). These data reveal a novel contribution of KCNQ gene products to the regulation of myogenic control of cerebral arterial diameter and suggest that Kv7 channel activating drugs may be appropriate candidates for the development of an effective therapy to ameliorate cerebral vasospasm.

Presenilin (PS) genes linked to early-onset familialAlzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of ␥-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/ KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Alzheimer's disease (AD)1 is a progressive dementing neurodegenerative disorder characterized by a massive deposition of ␤-amyloid and tau-rich neurofibrillary lesions in the brains (reviewed in Ref. 1 and references therein). A subset of AD is inherited as an autosomal dominant trait, and mutations in three different genes have thus far been linked to early-onset autosomal dominant forms of familial AD (FAD). Among these, presenilin 1 (PS1) and PS2 account for the majority of the early onset FAD (1). PS1 and PS2 genes encode polytopic integral membrane proteins that are predominantly localized in intracellular membranes and span the membrane six to eight times.PS proteins undergo endoproteolysis to give rise to N-and C-terminal fragments, which are the preponderant forms of endogenous PS in vivo (2). These fragments form a heterodimer and are incorporated into high molecular weight (HMW) protein complexes (2-5) that are highly stabilized (t1 ⁄2 ϭ ϳ20 h; Ref.6), whereas holoproteins of PS are rapidly degraded (t1 ⁄2 ϭ ϳ2 h) (6, 7). The steady-state levels of PS fragments seem to be tightly regulated by competition for shared, but limiting, cellular factors, because overexpression of PS in transfected cells does not increase the overall level of PS fragments and replaces endogenous PS (8).PS plays an important role in the generation of amyloid ␤ peptides (A␤) by facilitating intramembranous ␥-cleavage of ␤-amyloid protein precursor (␤APP), as evidenced by the lack of A␤ production and accumulation of ␤APP C-terminal stubs in cells established from PS-null mice (9 -11). In contrast, FADlinked mutations in PS increase the production of highly fibrillogenic A␤42 (12-15), which is the initial and predominantly deposited A␤ species in AD brains (16, 17) and normally consists of only ϳ10% of total secreted A␤ (18). Moreover, genetic studies in invertebrates and PS-null mice suggested that ␥-cleavage-like proteolytic cleavage at site 3 to release Notch intracellular domain (NICD), which is the prerequisite for Notch signaling (reviewed in Ref. 19), also is facilitated by PS. Furthermore, recent findings that the two intramembranous aspartates within the 6th and 7th transmembrane (TM) domains of PS are required for ␥-secretase activities (20) and that transition state analogue ␥-secretase inhibitors specifically label PS fragments (21-24) strongly support the notion that the PS-containing macroprotein complex catalyzes ␥-cleavage and that PS may represent the catalytic ...