Kirill Essin

Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

ABSTRACT 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.

Visceral Periadventitial Adipose Tissue Regulates Arterial Tone of Mesenteric Arteries

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (Kv) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 &mgr;mol/L), apamin (1 &mgr;mol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 &mgr;mol/L), or Ba2+ (3 &mgr;mol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via Kv channel activation in vascular smooth muscle cells.

Gq‐coupled receptors as mechanosensors mediating myogenic vasoconstriction

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 Gq/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 β‐arrestin. Agonist‐independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT1 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 AT1 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 Gq/11‐coupled receptors function as sensors of membrane stretch in VSM cells.

Pressure-induced and store-operated cation influx in vascular smooth muscle cells is independent of TRPC1

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 Ca2+ 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.

Hyperforin—a key constituent of St. John’s wort specifically activates TRPC6 channels

Hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, is the main active principle of St. Johns wort extract responsible for its anti‐depressive profile. Hyperforin inhibits the neuronal serotonin and norepinephrine uptake comparable to synthetic antidepressants. In contrast to synthetic anti‐depressants directly blocking neuronal amine uptake, hyperforin increases synaptic serotonin and norepi‐nephrine concentrations by an indirect and yet unknown mechanism. Our attempts to identify the molecular target of hyperforin resulted in the identification of TRPC6. Hyperforin induced sodium and calcium entry as well as currents in TRPC6‐expressing cells. Sodium currents and the subsequent breakdown of the membrane sodium gradients may be the rationale for the inhibition of neuronal amine uptake. The hyper‐forin‐induced cation entry was highly specific and related to TRPC6 and was suppressed in cells expressing a dominant negative mutant of TRPC6, whereas phylo‐genetically related channels, i.e., TRPC3 remained unaffected. Furthermore, hyperforin induces neuronal axonal sprouting like nerve growth factor in a TRPC6‐dependent manner. These findings support the role of TRPC channels in neurite extension and identify hyper‐forin as the first selective pharmacological tool to study TRPC6 function. Hyperforin integrates inhibition of neurotransmitter uptake and neurotrophic property by specific activation of TRPC6 and represents an interesting lead‐structure for a new class of antidepres‐sants.— Leuner, K., Kazanski, V., Müller, M., Essin, K., Henke, B., Gollasch, M., Harteneck, C., Müller, W. E. Hyperforin—a key constituent of St. Johns wort specifically activates TRPC6 channels. FASEB J. 21, 4101–4111 (2007)

Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation

The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18‐Epoxyeicosatetraenoic acid (17,18‐EETeTr) stimulates vascular large‐conductance K+ (BK) channels. BK channels are composed of the pore‐forming BK α and auxiliary BK β1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine‐sensitive calcium‐release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole‐cell and perforated‐patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague–Dawley rats, BK β1 gene‐deficient (−/−), BK α (−/−), RyR3 (−/−) and wild‐type mice. The 17,18‐EETeTr (100 nm) increased tetraethylammonium (1 mm)‐sensitive outward K+ currents in VSMC from wild‐type rats and wild‐type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15‐epoxyeicosa‐5(Z)‐enoic acid (10 μm). BK channel currents were increased 3.5‐fold in VSMC from BK β1 (−/−) mice, whereas a 2.9‐fold stimulation was observed in VSMC from RyR3 (−/−) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild‐type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca2+, 100 nm), nor by external calcium influx. The 17,18‐EETeTr did not induce outward currents in VSMC BK α (−/−) cells. We next tested the vasodilator effects of 17,18‐EETeTr on isolated arteries of BK α‐deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK α (−/−) mice compared with that observed in wild‐type and BK β1 (−/−) arteries. We conclude that 17,18‐EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18‐EETeTr is active in small arteries lacking BK β1, the data further suggest that BK α represents the molecular target for the principal action of 17,18‐EETeTr. Finally, the action of 17,18‐EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca2+ sparks stimulate nearby Ca2+‐activated K+ (BK) channels that hyperpolarize the membrane and close L‐type Ca2+ channels. We tested the contribution of L‐type Cav1.2 channels to Ca2+ spark regulation in tibial and cerebral artery VSMCs using VSMC‐specific Cav1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Cav1.2 channel gene inactivation reduced Ca2+ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca2+ levels and reduced sarcoplasmic reticulum (SR) Ca2+ load. Elevating cytosolic Ca2+ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild‐type and SMAKO VSMCs weakly reflected the voltage dependency of L‐type channels. This study provides evidence that local and tight coupling between the Cav1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca2+ sparks. Instead, Cav1.2 channels contribute to global cytosolic [Ca2+], which in turn influences luminal SR calcium and thus Ca2+ sparks.

Raloxifene Relaxes Rat Cerebral Arteries In Vitro and Inhibits L-Type Voltage-Sensitive Ca2+ Channels

Background and Purpose— Because of their mixed estrogen-agonist and estrogen-antagonist properties, selective estrogen receptor modulators (SERMs) are considered promising substitutes for hormone replacement therapy. Raloxifene and other SERMs confer estrogen-like cardiovascular protective effects but lack the carcinogenic activity of exogenous estrogen. However, little is known about the cerebrovascular action of raloxifene. Therefore, we studied the effects of raloxifene on the mechanisms regulating rat cerebral artery tone. Methods and Results— Ring segments of the isolated rat posterior communicating cerebral arteries were mounted in a microvessel myograph for measurement of isometric tension. Whole-cell L-type voltage-sensitive Ca2+ currents were recorded using the perforated patch-clamp technique. Raloxifene (0.1 to 10 μmol/L) reduced the contractile responses to U46619, phenylephrine, and endothelin-1 in normal Krebs solution or to CaCl2 in Ca2+-free, high K+-containing solution. Raloxifene-induced relaxation was identical in endothelium-intact and endothelium-denuded rings. ICI 182780 had no effect on raloxifene-induced relaxation. Raloxifene reduced L-type Ca2+ currents with a p D2 of 5.98±0.06, close to that (6.44±0.09) for raloxifene-induced relaxation of 60 mmol/L K+-contracted rings. Conclusions— This study demonstrates that raloxifene acutely relaxes rat cerebral arteries largely via an endothelium-independent mechanism, involving inhibition of Ca2+ influx through L-type Ca2+ channels.

BK channels in innate immune functions of neutrophils and macrophages

Oxygen-dependent antimicrobial activity of human polymorphonuclear leukocytes (PMNs) relies on the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate oxidants. As the oxidase transfers electrons from NADPH the membrane will depolarize and concomitantly terminate oxidase activity, unless there is charge translocation to compensate. Most experimental data implicate proton channels as the effectors of this charge compensation, although large-conductance Ca2+-activated K+ (BK) channels have been suggested to be essential for normal PMN antimicrobial activity. To test this latter notion, we directly assessed the role of BK channels in phagocyte function, including the NADPH oxidase. PMNs genetically lacking BK channels (BK(-/-)) had normal intracellular and extracellular NADPH oxidase activity in response to both receptor-independent and phagocytic challenges. Furthermore, NADPH oxidase activity of human PMNs and macrophages was normal after treatment with BK channel inhibitors. Although BK channel inhibitors suppressed endotoxin-mediated tumor necrosis factor-alpha secretion by bone marrow-derived macrophages (BMDMs), BMDMs of BK(-/-) and wild-type mice responded identically and exhibited the same ERK, PI3K/Akt, and nuclear factor-kappaB activation. Based on these data, we conclude that the BK channel is not required for NADPH oxidase activity in PMNs or macrophages or for endotoxin-triggered tumor necrosis factor-alpha release and signal transduction BMDMs.