Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of G q/11 -coupled receptors, which subsequently signal to TRPC channels in a G protein-and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of b-arrestin. Agonistindependent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT 1 angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT 1 receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that G q/11 -coupled receptors function as sensors of membrane stretch in VSM cells.

Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6 ؊/؊ mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6 ؊/؊ but not in WT mice. This effect was mirrored by a lack of hypoxiainduced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6 ؊/؊ mice. In both WT and TRPC6 ؊/؊ PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6 ؊/؊ PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.hypoxia-induced diacylglycerol accumulation ͉ precapillary pulmonary arterial smooth-muscle cells ͉ pulmonary hypertension ͉ transient receptor potential channel 6-deficient mouse model ͉ arterial hypoxemia A cute regional hypoxic pulmonary vasoconstriction (HPV) is necessary to maintain optimized gas exchange by directing blood flow from poorly ventilated to well ventilated areas of the lung. Under conditions of generalized hypoxia, however, total pulmonary vascular resistance rises with subsequent increase of right heart load (1-3). Chronic hypoxia, as occurring in ventilatory disorders induces chronic pulmonary hypertension, pulmonary vascular remodeling, and cor pulmonale (4). The underlying oxygen sensing and signal transduction mechanisms of the acute and chronic vascular responses are largely unknown. A rise of intracellular calcium ([Ca 2ϩ ] i ) in pulmonary artery smooth-muscle cells (SMCs) has been suggested to be the key event in these processes (5-8). However, the question how [Ca 2ϩ ] i is regulated has not yet been resolved. Among others, transient receptor potential (TRP) channels are regulators of [Ca 2ϩ ] i . The TRP protein superfamily consists of a diverse group of nonselective cation channels involved in many basic cellular processes (9). Whereas members of the TRPV and TRPM subfamilies have emerged as versatile cellular sensors, the functional importance of the seven members (TRPC1 to -7) of the TRPC (transient receptor potential cation channel subfamily C) subfamily...

Coordinated release of calcium (Ca2+) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptor (RYR2) channels is essential for cardiomyocyte function. In catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disease characterized by stress-induced ventricular arrhythmias in young patients with structurally normal hearts, autosomal dominant mutations in RYR2 or recessive mutations in calsequestrin lead to aberrant diastolic Ca2+ release from the SR causing arrhythmogenic delayed after depolarizations (DADs). Here, we report the generation of induced pluripotent stem cells (iPSCs) from a CPVT patient carrying a novel RYR2 S406L mutation. In patient iPSC-derived cardiomyocytes, catecholaminergic stress led to elevated diastolic Ca2+ concentrations, a reduced SR Ca2+ content and an increased susceptibility to DADs and arrhythmia as compared to control myocytes. This was due to increased frequency and duration of elementary Ca2+ release events (Ca2+ sparks). Dantrolene, a drug effective on malignant hyperthermia, restored normal Ca2+ spark properties and rescued the arrhythmogenic phenotype. This suggests defective inter-domain interactions within the RYR2 channel as the pathomechanism of the S406L mutation. Our work provides a new in vitro model to study the pathogenesis of human cardiac arrhythmias and develop novel therapies for CPVT.

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 ؊/؊ smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 ؊/؊ smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.The TRP (transient receptor potential) family of ion channels is a growing group of structurally and evolutionarily related cation channels formed of several subfamilies that include the TRPC, TRPM, and TRPV classes of channels (6, 22). TRP-type ion channels are presumed to be homo-or heterotetramers (13,14), each spanning the plasma membrane six times. The founding members of this channel family are the insect TRP and TRPL channels, which are responsible for photoreceptor depolarization in response to light. Mammalian TRPCs (C stands for canonical or classical) (23, 32) are the closest mammalian structural relatives of insect TRPs. Among the TRPC channels, TRPC3, -6, and -7 are 75% identical and gated by signal transduction pathways that activate C-type phospholipases (3, 32) as well as by direct exposure to diacylglycerols (DAG) (15). TRPC3, -6, and -7 interact physically and, upon coexpression, coassemble to form functional channels (14). Expression of TRPC3 and TRPC7 in HEK 293 cells, but not of TRPC6, reveals constitutively active cation channels that are permeable not only to monovalent but also to divalent cations such as Ca 2ϩ , Ba 2ϩ , and Mn 2ϩ (7,23,33). In contrast to members of other TRP families, the functional importance of most members of the TRPC subfamily is still poorly understood. A TRPC channel for which considerable evidence has accumulated for a specific role is TRPC6, which has been proposed to regulate smooth muscle function. The TRPC6 mRNA was originally isolated from mouse brain and was also identified in lung cells (4). By comparative biophysical characterization and gene suppression using antisense oligonucleot...

Among the classical transient receptor potential (TRPC) subfamily, TRPC1 is described as a mechanosensitive and store-operated channel proposed to be activated by hypoosmotic cell swelling and positive pipette pressure as well as regulated by the filling status of intracellular Ca(2+) stores. However, evidence for a physiological role of TRPC1 may most compellingly be obtained by the analysis of a TRPC1-deficient mouse model. Therefore, we have developed and analyzed TRPC1(-/-) mice. Pressure-induced constriction of cerebral arteries was not impaired in TRPC1(-/-) mice. Smooth muscle cells from cerebral arteries activated by hypoosmotic swelling and positive pipette pressure showed no significant differences in cation currents compared to wild-type cells. Moreover, smooth muscle cells of TRPC1(-/-) mice isolated from thoracic aortas and cerebral arteries showed no change in store-operated cation influx induced by thapsigargin, inositol-1,4,5 trisphosphate, and cyclopiazonic acid compared to cells from wild-type mice. In contrast to these results, small interference RNAs decreasing the expression of stromal interaction molecule 1 (STIM1) inhibited thapsigargin-induced store-operated cation influx, demonstrating that STIM1 and TRPC1 are mutually independent. These findings also imply that, as opposed to current concepts, TRPC1 is not an obligatory component of store-operated and stretch-activated ion channel complexes in vascular smooth muscle cells.