AXL receptor tyrosine kinase modulates gonadotropin-releasing hormone receptor signaling.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) receptors are essential for reproduction and are expressed in numerous urogenital, reproductive, and non-reproductive cancers. In addition to canonical G protein-coupled receptor signaling, GnRH receptors functionally interact with several receptor tyrosine kinases. AXL is a receptor tyrosine kinase expressed in numerous tissues as well as multiple tumors. Here we tested the hypothesis that AXL, along with its endogenous ligand Gas6, impacts GnRH receptor signaling. METHODS: We used clonal murine pituitary αT3-1 and LßT2 gonadotrope cell lines to examine the effect of AXL activation on GnRH receptor-dependent signaling outcomes. ELISA and immunofluorescence were used to observe AXL and GnRH receptor expression in αT3-1 and LßT2 cells, as well as in murine and human pituitary sections. We also used ELISA to measure changes in ERK phosphorylation, pro-MMP9 production, and release of LHß. Digital droplet PCR was used to measure the abundance of Egr-1 transcripts. A transwell migration assay was used to measure αT3-1 and LßT2 migration responses to GnRH and AXL. RESULTS: We observed AXL, along with the GnRH receptor, expression in αT3-1 and LßT2 gonadotrope cell lines, as well as in murine and human pituitary sections. Consistent with a potentiating role of AXL, Gas6 enhanced GnRH-dependent ERK phosphorylation in αT3-1 and LßT2 cells. Further, and consistent with enhanced post-transcriptional GnRH receptor responses, we found that Gas6 increased the abundance of Egr-1 transcripts. Suggesting functional significance, in LßT2 cells, Gas6/AXL signaling stimulated LHß production and enhanced GnRH receptor-dependent generation of pro-MMP9 protein and promoted cell migration. CONCLUSIONS: Altogether, these data describe a novel role for AXL as a modulator of GnRH receptor signaling. Video Abstract.

AXL/Gas6 signaling mechanisms in the hypothalamic-pituitary-gonadal axis.

AXL is a receptor tyrosine kinase commonly associated with a variety of human cancers. Along with its ligand Gas6 (growth arrest-specific protein 6), AXL is emerging as an important regulator of neuroendocrine development and function. AXL signaling in response to Gas6 binding impacts neuroendocrine structure and function at the level of the brain, pituitary, and gonads. During development, AXL has been identified as an upstream inhibitor of gonadotropin receptor hormone (GnRH) production and also plays a key role in the migration of GnRH neurons from the olfactory placode to the forebrain. AXL is implicated in reproductive diseases including some forms of idiopathic hypogonadotropic hypogonadism and evidence suggests that AXL is required for normal spermatogenesis. Here, we highlight research describing AXL/Gas6 signaling mechanisms with a focus on the molecular pathways related to neuroendocrine function in health and disease. In doing so, we aim to present a concise account of known AXL/Gas6 signaling mechanisms to identify current knowledge gaps and inspire future research.

Characterization of the A-type potassium current in murine gastric fundus smooth muscles.

Transient outward, or "A-type," currents are rapidly inactivating voltage-gated potassium currents that operate at negative membrane potentials. A-type currents have not been reported in the gastric fundus, a tonic smooth muscle. We used whole cell voltage clamp to identify and characterize A-type currents in smooth muscle cells (SMCs) isolated from murine fundus. A-type currents were robust in these cells with peak amplitudes averaging 1.5 nA at 0 mV. Inactivation was rapid with a time constant of 71 ms at 0 mV; recovery from inactivation at -80 mV was similarly rapid with a time constant of 75 ms. A-type currents in fundus were blocked by 4-aminopyridine (4-AP), flecainide, and phrixotoxin-1 (PaTX1). Remaining currents after 4-AP and PaTX1 displayed half-activation potentials that were shifted to more positive potentials and showed incomplete inactivation. Currents after tetraethylammonium (TEA) displayed half inactivation at -48.1 ± 1.0 mV. Conventional microelectrode and contractile experiments on intact fundus muscles showed that 4-AP depolarized membrane potential and increased tone under conditions in which enteric neurotransmission was blocked. These data suggest that A-type K+ channels in fundus SMCs are likely active at physiological membrane potentials, and sustained activation of A-type channels contributes to the negative membrane potentials of this tonic smooth muscle. Quantitative analysis of Kv4 expression showed that Kcnd3 was dominantly expressed in fundus SMCs. These data were confirmed by immunohistochemistry, which revealed Kv4.3-like immunoreactivity within the tunica muscularis. These observations indicate that Kv4 channels likely form the A-type current in murine fundus SMCs.

Angiotensin-II Modulates GABAergic Neurotransmission in the Mouse Substantia Nigra.

GABAergic projections neurons of the substantia nigra reticulata (SNr), through an extensive network of dendritic arbors and axon collaterals, provide robust inhibitory input to neighboring dopaminergic neurons in the substantia nigra compacta (SNc). Angiotensin-II (Ang-II) receptor signaling increases SNc dopaminergic neuronal sensitivity to insult, thus rendering these cells susceptible to dysfunction and destruction. However, the mechanisms by which Ang-II regulates SNc dopaminergic neuronal activity are unclear. Given the complex relationship between SN dopaminergic and GABAergic neurons, we hypothesized that Ang-II could regulate SNc dopaminergic neuronal activity directly and indirectly by modulating SNr GABAergic neurotransmission. Here, using transgenic mice, slice electrophysiology, and optogenetics, we provide evidence of an AT1 receptor-mediated signaling mechanism in SNr GABAergic neurons where Ang-II suppresses electrically-evoked neuronal output by facilitating postsynaptic GABAA receptors (GABAARs) and prolonging the action potential (AP) duration. Unexpectedly, Ang-II had no discernable effects on the electrical properties of SNc dopaminergic neurons. Also, and indicating a nonlinear relationship between electrical activity and neuronal output, following phasic photoactivation of SNr GABAergic neurons, Ang-II paradoxically enhanced the feedforward inhibitory input to SNc dopaminergic neurons. In sum, our observations describe an increasingly complex and heterogeneous response of the SN to Ang-II by revealing cell-specific responses and nonlinear effects on intranigral GABAergic neurotransmission. Our data further implicate the renin-angiotensin-system (RAS) as a functionally relevant neuromodulator in the substantia nigra, thus underscoring a need for additional inquiry.

Reactive Oxygen Species Modulate Activity-Dependent AMPA Receptor Transport in C. elegans.

The AMPA subtype of synaptic glutamate receptors (AMPARs) plays an essential role in cognition. Their function, numbers, and change at synapses during synaptic plasticity are tightly regulated by neuronal activity. Although we know that long-distance transport of AMPARs is essential for this regulation, we do not understand the associated regulatory mechanisms of it. Neuronal transmission is a metabolically demanding process in which ATP consumption and production are tightly coupled and regulated. Aerobic ATP synthesis unavoidably produces reactive oxygen species (ROS), such as hydrogen peroxide, which are known modulators of calcium signaling. Although a role for calcium signaling in AMPAR transport has been described, there is little understanding of the mechanisms involved and no known link to physiological ROS signaling. Here, using real-time in vivo imaging of AMPAR transport in the intact C. elegans nervous system, we demonstrate that long-distance synaptic AMPAR transport is bidirectionally regulated by calcium influx and activation of calcium/calmodulin-dependent protein kinase II. Quantification of in vivo calcium dynamics revealed that modest, physiological increases in ROS decrease calcium transients in C. elegans glutamatergic neurons. By combining genetic and pharmacological manipulation of ROS levels and calcium influx, we reveal a mechanism in which physiological increases in ROS cause a decrease in synaptic AMPAR transport and delivery by modulating activity-dependent calcium signaling. Together, our results identify a novel role for oxidant signaling in the regulation of synaptic AMPAR transport and delivery, which in turn could be critical for coupling the metabolic demands of neuronal activity with excitatory neurotransmission.SIGNIFICANCE STATEMENT Synaptic AMPARs are critical for excitatory synaptic transmission. The disruption of their synaptic localization and numbers is associated with numerous psychiatric, neurologic, and neurodegenerative conditions. However, very little is known about the regulatory mechanisms controlling transport and delivery of AMPAR to synapses. Here, we describe a novel physiological signaling mechanism in which ROS, such as hydrogen peroxide, modulate AMPAR transport by modifying activity-dependent calcium signaling. Our findings provide the first evidence in support of a mechanistic link between physiological ROS signaling, AMPAR transport, localization, and excitatory transmission. This is of fundamental and clinical significance since dysregulation of intracellular calcium and ROS signaling is implicated in aging and the pathogenesis of several neurodegenerative disorders, including Alzheimer's and Parkinson's disease.

Subplasmalemmal hydrogen peroxide triggers calcium influx in gonadotropes.

Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells (gonadotropes) initiates multiple signaling cascades that culminate in the secretion of luteinizing and follicle-stimulating hormones, which have critical roles in fertility and reproduction. Enhanced luteinizing hormone biosynthesis, a necessary event for ovulation, requires a signaling pathway characterized by calcium influx through L-type calcium channels and subsequent activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK). We previously reported that highly localized subplasmalemmal calcium microdomains produced by L-type calcium channels (calcium sparklets) play an essential part in GnRH-dependent ERK activation. Similar to calcium, reactive oxygen species (ROS) are ubiquitous intracellular signaling molecules whose subcellular localization determines their specificity. To investigate the potential influence of oxidant signaling in gonadotropes, here we examined the impact of ROS generation on L-type calcium channel function. Total internal reflection fluorescence (TIRF) microscopy revealed that GnRH induces spatially restricted sites of ROS generation in gonadotrope-derived αT3-1 cells. Furthermore, GnRH-dependent stimulation of L-type calcium channels required intracellular hydrogen peroxide signaling in these cells and in primary mouse gonadotropes. NADPH oxidase and mitochondrial ROS generation were each necessary for GnRH-mediated stimulation of L-type calcium channels. Congruently, GnRH increased oxidation within subplasmalemmal mitochondria, and L-type calcium channel activity correlated strongly with the presence of adjacent mitochondria. Collectively, our results provide compelling evidence that NADPH oxidase activity and mitochondria-derived hydrogen peroxide signaling play a fundamental role in GnRH-dependent stimulation of L-type calcium channels in anterior pituitary gonadotropes.

Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK.

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

Arterial Smooth Muscle Mitochondria Amplify Hydrogen Peroxide Microdomains Functionally Coupled to L-Type Calcium Channels.

RATIONALE: Mitochondria are key integrators of convergent intracellular signaling pathways. Two important second messengers modulated by mitochondria are calcium and reactive oxygen species. To date, coherent mechanisms describing mitochondrial integration of calcium and oxidative signaling in arterial smooth muscle are incomplete. OBJECTIVE: To address and add clarity to this issue, we tested the hypothesis that mitochondria regulate subplasmalemmal calcium and hydrogen peroxide microdomain signaling in cerebral arterial smooth muscle. METHODS AND RESULTS: Using an image-based approach, we investigated the impact of mitochondrial regulation of L-type calcium channels on subcellular calcium and reactive oxygen species signaling microdomains in isolated arterial smooth muscle cells. Our single-cell observations were then related experimentally to intact arterial segments and to living animals. We found that subplasmalemmal mitochondrial amplification of hydrogen peroxide microdomain signaling stimulates L-type calcium channels, and that this mechanism strongly impacts the functional capacity of the vasoconstrictor angiotensin II. Importantly, we also found that disrupting this mitochondrial amplification mechanism in vivo normalized arterial function and attenuated the hypertensive response to systemic endothelial dysfunction. CONCLUSIONS: From these observations, we conclude that mitochondrial amplification of subplasmalemmal calcium and hydrogen peroxide microdomain signaling is a fundamental mechanism regulating arterial smooth muscle function. As the principle components involved are fairly ubiquitous and positioning of mitochondria near the plasma membrane is not restricted to arterial smooth muscle, this mechanism could occur in many cell types and contribute to pathological elevations of intracellular calcium and increased oxidative stress associated with many diseases.

GnRH evokes localized subplasmalemmal calcium signaling in gonadotropes.

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.

Calcium dynamics in vascular smooth muscle.

Smooth muscle cells are ultimately responsible for determining vascular luminal diameter and blood flow. Dynamic changes in intracellular calcium are a critical mechanism regulating vascular smooth muscle contractility. Processes influencing intracellular calcium are therefore important regulators of vascular function with physiological and pathophysiological consequences. In this review we discuss the major dynamic calcium signals identified and characterized in vascular smooth muscle cells. These signals vary with respect to their mechanisms of generation, temporal properties, and spatial distributions. The calcium signals discussed include calcium waves, junctional calcium transients, calcium sparks, calcium puffs, and L-type calcium channel sparklets. For each calcium signal we address underlying mechanisms, general properties, physiological importance, and regulation.

Local regulation of L-type Ca²âº channel sparklets in arterial smooth muscle.

This review addresses the latest advances in our understanding of the regulation of a novel Ca²âº signal called L-type Ca²âº channel sparklets in arterial smooth muscle. L-type Ca²âº channel sparklets are elementary Ca²âº influx events produced by the opening of a single or a small cluster of L-type Ca²âº channels. These Ca²âº signals were first visualized in the vasculature in arterial smooth muscle cells. In these cells, L-type Ca²âº channel sparklets display two functionally distinct gating modalities that regulate local and global [Ca²âº](i). The activity of L-type Ca²âº channel sparklets varies regionally within a cell depending on the dynamic activity of a cohort of protein kinases and phosphatases recruited to L-type Ca²âº channels in the arterial smooth muscle sarcolemma in a complex coordinated by the scaffolding molecule AKAP150. We also described a mechanism whereby clusters of L-type Ca²âº channels gate cooperatively to amplify intracellular Ca²âº signals with likely pathological consequences.

Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C.

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.

Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels.

Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.

Pharmacological inhibition of TRPM4 hyperpolarizes vascular smooth muscle.

The contractile state of vascular smooth muscle cells is regulated by small changes in membrane potential that gate voltage-dependent calcium channels. The melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure-induced membrane depolarization and arterial constriction. A recent study shows that the tricyclic compound 9-phenanthrol inhibits TRPM4, but not the related channel TRPM5. The current study investigated the specificity of 9-phenanthrol and the effects of the compound on pressure-induced smooth muscle depolarization and arterial constriction. Patch-clamp electrophysiology revealed that 9-phenanthrol blocks native TRPM4 currents in freshly isolated smooth muscle cells in a concentration-dependent manner (IC(50) = 10.6 µM). 9-Phenanthrol (30 µM) had no effect on maximum evoked currents in human embryonic kidney cells expressing recombinant TRPC3 or TRPC6 channels. Large-conductance Ca(2+)-activated K(+), voltage-dependent K(+), inwardly rectifying K(+), and voltage-dependent Ca(2+) channel activity in native cerebral artery myocytes was not altered by administration of 9-phenanthrol (30 µM). Using intracellular microelectrodes to record smooth muscle membrane potential in isolated cerebral arteries pressurized to 70 mmHg, we found that 9-phenanthrol (30 µM) reversibly hyperpolarized the membrane from ∼-40 mV to ∼-70 mV. In addition, we found that myogenic tone was reversibly abolished when vessels were exposed to 9-phenanthrol. These data demonstrate that 9-phenanthrol is useful for studying the functional significance of TRPM4 in vascular smooth muscle cells and that TRPM4 is an important regulator of smooth muscle cell membrane depolarization and arterial constriction in response to intraluminal pressure.

Local regulation of arterial L-type calcium channels by reactive oxygen species.

RATIONALE: Reactive oxygen species (ROS) are implicated in the development of cardiovascular disease, and oxidants are important signaling molecules in many cell types. Recent evidence suggests that localized subcellular compartmentalization of ROS generation is an important feature of ROS signaling. However, mechanisms that transduce localized subcellular changes in redox status to functionally relevant changes in cellular processes such as Ca(2+) influx are poorly understood. OBJECTIVE: To test the hypothesis that ROS regulate L-type Ca(2+) channel activity in cerebral arterial smooth muscle. METHODS AND RESULTS: Using a total internal reflection fluorescence imaging-based approach, we found that highly localized subplasmalemmal generation of endogenous ROS preceded and colocalized with sites of enhanced L-type Ca(2+) channel sparklet activity in isolated cerebral arterial smooth muscle cells. Consistent with this observation and our hypothesis, exogenous ROS increased localized L-type Ca(2+) channel sparklet activity in isolated arterial myocytes via activation of protein kinase Cα and when applied to intact cerebral arterial segments, exogenous ROS increased arterial tone in an L-type Ca(2+) channel-dependent fashion. Furthermore, angiotensin II-dependent stimulation of local L-type Ca(2+) channel sparklet activity in isolated cells and contraction of intact arteries was abolished following inhibition of NADPH oxidase. CONCLUSIONS: Our data support a novel model of local oxidative regulation of Ca(2+) influx where vasoconstrictors coupled to NAPDH oxidase (eg, angiotensin II) induce discrete sites of ROS generation resulting in oxidative activation of adjacent protein kinase Cα molecules that in turn promote local sites of enhanced L-type Ca(2+) channel activity, resulting in increased Ca(2+) influx and contraction.

Vasoconstriction resulting from dynamic membrane trafficking of TRPM4 in vascular smooth muscle cells.

The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 +/- 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (approximately 3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (approximately 3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCalpha and PKCbeta activity but was inhibited by blockade of PKCdelta with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCdelta. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCdelta. We conclude that PKCdelta activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

Ca2+ release from the sarcoplasmic reticulum is required for sustained TRPM4 activity in cerebral artery smooth muscle cells.

The melastatin transient receptor potential (TRP) channel TRPM4 is a critical regulator of vascular smooth muscle cell membrane potential and contractility. Activation of the channel is Ca(2+)-dependent, but prolonged exposure to high (>1 microM) levels of intracellular Ca(2+) causes rapid (within approximately 2 min) desensitization of TRPM4 currents under conventional whole cell and inside-out patch-clamp conditions. The goal of the present study was to establish a novel method to record sustained TRPM4 currents in smooth muscle cells under near-physiological conditions. Using the amphotericin B-perforated patch-clamp technique, we recorded and characterized sustained (up to 30 min) transient inward cation currents (TICCs) in freshly isolated cerebral artery myocytes. In symmetrical cation solutions, TICCs reversed at 0 mV and had an apparent unitary conductance of 25 pS. Replacement of extracellular Na(+) with the nonpermeable cation N-methyl-d-glucamine abolished the current. TICC activity was attenuated by the TRPM4 blockers fluflenamic acid and 9-phenanthrol. Selective silencing of TRPM4 expression using small interfering RNA diminished TICC activity, suggesting that the molecular identity of the responsible ion channel is TRPM4. We used the perforated patch-clamp method to test the hypothesis that TRPM4 is activated by intracellular Ca(2+) signaling events. We found that TICC activity is independent of Ca(2+) influx and ryanodine receptor activity but is attenuated by sarco(endo)plasmic reticulum Ca(2+)-ATPase inhibition and blockade of inositol 1,4,5-trisphosphate receptor-mediated Ca(2+) release from the sarcoplasmic reticulum. Our findings suggest that TRPM4 channels in cerebral artery myocytes are regulated by Ca(2+) release from inositol 1,4,5-trisphosphate receptor on the sarcoplasmic reticulum.

The control of Ca2+ influx and NFATc3 signaling in arterial smooth muscle during hypertension.

Many excitable cells express L-type Ca(2+) channels (LTCCs), which participate in physiological and pathophysiological processes ranging from memory, secretion, and contraction to epilepsy, heart failure, and hypertension. Clusters of LTCCs can operate in a PKCalpha-dependent, high open probability mode that generates sites of sustained Ca(2+) influx called "persistent Ca(2+) sparklets." Although increased LTCC activity is necessary for the development of vascular dysfunction during hypertension, the mechanisms leading to increased LTCC function are unclear. Here, we tested the hypothesis that increased PKCalpha and persistent Ca(2+) sparklet activity contributes to arterial dysfunction during hypertension. We found that PKCalpha and persistent Ca(2+) sparklet activity is indeed increased in arterial myocytes during hypertension. Furthermore, in human arterial myocytes, PKCalpha-dependent persistent Ca(2+) sparklets activated the prohypertensive calcineurin/NFATc3 signaling cascade. These events culminated in three hallmark signs of hypertension-associated vascular dysfunction: increased Ca(2+) entry, elevated arterial [Ca(2+)](i), and enhanced myogenic tone. Consistent with these observations, we show that PKCalpha ablation is protective against the development of angiotensin II-induced hypertension. These data support a model in which persistent Ca(2+) sparklets, PKCalpha, and calcineurin form a subcellular signaling triad controlling NFATc3-dependent gene expression, arterial function, and blood pressure. Because of the ubiquity of these proteins, this model may represent a general signaling pathway controlling gene expression and cellular function.

Calcium sparklets in arterial smooth muscle.

Voltage-dependent, L-type Ca2+ channels (LTCC) play an essential role in arterial smooth muscle contraction and, consequently, the regulation of arterial diameter, tissue perfusion and blood pressure. However, the spatial organization of functional LTCC in arterial myocytes is incompletely understood. Total internal reflection fluorescence and swept-field confocal microscopy revealed that the opening of a single or a cluster of LTCC produces local elevations in [Ca2+]i called Ca2+ sparklets. In arterial myocytes, Ca2+ sparklets are produced by the opening of Cav1.2 channels. The Ca2+ sparklet activity is bimodal. In low activity mode, rare stochastic openings of solitary LTCC produce limited Ca2+ influx ('low activity Ca2+ sparklets'). In contrast, discrete clusters of LTCC associated with protein kinase Ca (PKCa) operate in a sustained, high-activity mode resulting in substantial Ca2+ influx ('persistent Ca2+ sparklets'). The Ca2+ sparklet activity varies regionally within a myocyte depending on the relative activities of nearby PKCa and opposing protein phosphates 2A and 2B. Low- and high-activity persistent Ca2+ sparklets modulate local and global [Ca2+]i in arterial smooth muscle, suggesting that this Ca2+ signal may play an important role in the regulation of vascular function.