Arteriolar Smooth Muscle Research Articles

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and PurposeCa2+ imaging reveals subcellular Ca2+ sparks and global Ca2+ waves/oscillations in vascular smooth muscle. It is well established that Ca2+ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca2+ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca2+ sparks in the generation of myogenic tone in pressurized arterioles.Experimental ApproachIsolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca2+ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.Key ResultsTone development was associated with an increased frequency of Ca2+ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca2+ oscillations, quantifiable as an increased cross‐correlation coefficient. Inhibition of Ca2+ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca2+, resulted in arteriolar relaxation. Cyclopiazonic acid‐induced dilatation was associated with decreased Ca2+ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca2+] ([Ca2+]c), as measured using Fura2 and microfluorimetry.Conclusions and ImplicationsThis study provides direct evidence that Ca2+ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca2+]c, underlining the importance of frequency‐modulated signalling in vascular smooth muscle.

Fenamates block gap junction coupling and potentiate BKCa channels in guinea pig arteriolar cells

We determined the actions of the fenamates, flufenamic acid (FFA) and niflumic acid (NFA), on gap junction-mediated intercellular coupling between vascular smooth muscle cells (VSMC) in situ of acutely isolated arteriole segments from the three vascular beds: the spiral modiolar artery (SMA), anterior inferior cerebellar artery (AICA) and mesenteric artery (MA), and on non-junctional membrane channels in dispersed VSMCs. Conventional whole-cell recording methods were used. FFA reversibly suppressed the input conductance (Ginput) or increased the input resistance (Rinput) in a concentration dependent manner, with slightly different IC50s for the SMA, AICA and MA segments (26, 33 and 56μM respectively, P>0.05). Complete electrical isolation of the recorded VSMC was normally reached at ≥300μM. NFA had a similar effect on gap junction among VSMCs with an IC50 of 40, 48 and 62μM in SMA, AICA and MA segments, respectively. In dispersed VSMCs, FFA and NFA increased outward rectifier K+-current mediated by the big conductance calcium-activated potassium channel (BKCa) in a concentration-dependent manner, with a similar EC50 of ∼300μM for both FFA and NFA in the three vessels. Iberiotoxin, a selective blocker of the BKCa, suppressed the enhancement of the BKCa by FFA and NFA. The KV blocker 4-AP had no effect on the fenamates-induced K+-current enhancement. We conclude that FFA and NFA blocked the vascular gap junction mediated electrical couplings uniformly in arterioles of the three vascular beds, and complete electrical isolation of the recorded VSMC is obtained at ≧300μM; FFA and NFA also activate BKCa channels in the arteriolar smooth muscle cells in addition to their known inhibitory effects on chloride channels.

Mechanisms underlying regional differences in the Ca2+ sensitivity of BKCa current in arteriolar smooth muscle

Abstract β1-Subunits enhance the gating properties of large-conductance Ca(2+)-activated K(+) channels (BKCa) formed by α-subunits. In arterial vascular smooth muscle cells (VSMCs), β1-subunits are vital in coupling SR-generated Ca(2+) sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional β1-subunit:α-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. β1 modulation of the α-subunit Ca(2+) sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca(2+) was raised from 0.5 to 100 μm. Calculated V1/2 values of channel activation decreased from 72.0 ± 6.1 at 0.5 μm Ca(2+)i to -89 ± 9 mV at 100 μm Ca(2+)i in cerebral compared with 101 ± 10 to -63 ± 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an ∼2.5-fold weaker apparent Ca(2+) sensitivity such that at a value of Vm of -30 mV, a mean value of [Ca(2+)]i of 39 μm was required to open half of the channels in cremaster versus 16 μm [Ca(2+)]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either -30 or 30 mV at any given [Ca(2+)]. β1-Subunit-directed siRNA decreased both the apparent Ca(2+) sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of β1-subunit:α-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca(2+) sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.

Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup.

Mutations of both nuclear and mitochondrial DNA (mtDNA)-encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator-1. Ten homozygous null (adenine nucleotide translocator-1(-/-)) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H.

Nitric Oxide Can Directly Mediate Renin Cell Recruitment

See related article, pp 400–407 In this issue of Hypertension , Neubauer et al1 fit missing pieces into the puzzle of how renin production is controlled, when the demand for renin is high. Our understanding of the acute release of renin is much more complete than what we know about the long-term adaptations to stimuli of renin. Our scarce knowledge of the mechanisms mediating long-term renin stimulation is remarkable with regard to the clinical importance of renin. Hypertension, fluid and electrolyte homeostasis, as well as inflammation and fibrosis all may involve chronic renin stimulation.2 More than 400 million years ago, renin first appeared in organisms,3 and renin is often seen as the first hormone discovered. Shortly before the 20th century, Tigerstedt and Bergman showed that kidney extracts can elevate blood pressure, when intravenously infused.4 Today, we recognize that the actions of renin go far beyond that of controlling blood pressure, and we have detailed insight into how renin is released into the circulation.5 The major stimuli of short-term renin release are blood pressure decreases, β1-adrenergic stimulation, and low salt, whereas …

Mechanism of spironolactone-induced Ca<sup>2+</sup> increase in rat testicular arteriole smooth muscle cells revealed by real-time laser scanning confocal microscopy

Summary. We reported previously that spironolactone (SPL) induced an increase in intracellular Ca 2+ concentration ([Ca 2+ ]i) in rat testicular arteriole smooth muscle cells. In the present study, we further investigated the mechanism of SPL-induced [Ca 2+ ]i dynamics in rat arteriole smooth muscles. The increase in [Ca 2+ ]i induced by SPL (300 μM) was markedly inhibited in extracellular Ca 2+ -free conditions and in

Temporal Development of Retinal Arteriolar Endothelial Dysfunction in Porcine Type 1 Diabetes

Although hyperglycemia is implicated in retinal vascular dysfunction associated with the development of diabetic retinopathy, the temporal influence of hyperglycemia on retinal arteriolar reactivity remains unclear. Development of a large animal model of diabetes relevant to the human retina for evaluation of vascular function is also lacking. Herein, we examined nitric oxide (NO)-mediated dilation and endothelin-1 (ET-1)-induced constriction in retinal arterioles at various time periods in a porcine model of type 1 diabetes. Retinal arterioles were isolated from streptozocin-induced diabetic pigs (2, 6, and 12 weeks of hyperglycemia, 427 ± 23 mg/dL) and age-matched control pigs (73 ± 4 mg/dL), and then cannulated and pressurized for vasoreactivity study using videomicroscopic techniques. Retinal arterioles isolated from control and diabetic pigs developed comparable levels of myogenic tone. The endothelium-dependent NO-mediated vasodilations to bradykinin and stepwise increases in luminal flow were significantly reduced within 2 weeks of hyperglycemia. The inhibitory effect was comparable following 6 and 12 weeks of hyperglycemia. However, the endothelium-independent vasodilation to sodium nitroprusside was unaffected. Constriction of retinal arterioles to ET-1 was unaltered at all time periods of hyperglycemia. Our findings provide the first direct evidence for selective impairment of endothelium-dependent NO-mediated dilation of retinal arterioles within 2 weeks of hyperglycemia in a pig model of diabetes. By contrast, the ability of arteriolar smooth muscle to dilate to NO donor or contract to ET-1 was unaffected throughout the study period. This endothelial vasodilator dysfunction during early diabetes may contribute to development of retinopathy with chronic hyperglycemia.

Effects of sympathetic blockade on pressure dependent abdominal aortic stiffness in the spontaneously hypertensive rat

Introduction • Increased sympathetic nerve activity (SNA) is associated with development of hypertension in spontaneously hypertensive rats (SHR). • Elevated SNA increases arteriolar smooth muscle tone affecting peripheral resistance, however, the effect on large arteries is not well established. • This study aimed to assesses the effect of SNA on aortic stiffness in SHR’s using sympathetic blockade at different values of mean arterial pressure (MAP). Results PWV

English

Dyslipidemia and oxidative stress were evaluated in patients with polycystic ovarian syndrome. MATERIALS AND METHODS: Total cholesterol, Triglyceride, HDL cholesterol, LDL cholesterol, Malondialdehyde (MDA) and Total antioxidant capacity were measured in serum of PCOS subjects and age matched controls. RESULTS: Study group comprised of 31 women with PCOS and control group with 31 healthy volunteers. Mean serum levels of MDA, Cholesterol, Triglycerides and LDL cholesterol were significantly increased and TAC and HDL cholesterol were significantly decreased in PCOS subjects compared to controls. CONCLUSION: Our results revealed that PCOS is associated with dyslipidemia and altered oxidative status. Women with PCOS would be predicted to be at high risk for dyslipidemia because they have elevated androgen levels and are frequently obese. Moreover, since they are also often hyperinsulinemic and insulin resistant, they would also be expected to be at increased risk for the dyslipidemia associated with insulin resistance. Insulin, rather than androgen, levels correlate best with lipid abnormalities, and suppressing androgen levels does not alter lipid profiles in PCOS. Insulin resistance and hyperinsulinemia are also associated with an atherogenic plasma lipid profile. Elevated plasma insulin concentrations enhance very low density lipoprotein (VLDL) synthesis, leading to hypertriglyceridemia. Progressive elimination of lipid and apolipoproteins from the VLDL particle leads to an increased formation of intermediate-density and low-density lipoproteins, both of which are atherogenic. Last, insulin, independent of its effects on blood pressure and plasma lipids, is known to be atherogenic. The hormone enhances cholesterol transport into arteriolar smooth muscle cells and increases

Pharmacological Profiling of Store‐Operated Ca2+ Entry in Retinal Arteriolar Smooth Muscle

Pharmacological profiling of SOCE and molecular profiling of ORAI and TRPC expression in arterioles. Fura-2-based microfluorimetry was used to assess CPA-induced SOCE in rat retinal arteriolar myocytes. Arteriolar ORAI and TRP transcript expression was screened using RT-PCR. The SKF96365 and LOE908 blocked SOCE (IC(50) s of 1.2 and 1.4 μm, respectively). Gd(3+) and La(3+) potently inhibited SOCE (IC(50) s of 21 and 42 nm, respectively), but Ni(2+) showed lower potency (IC(50) = 11.6 μm). 2APB inhibited SOCE (IC(50) = 3.7 μm) but enhanced basal influx (>100 μm). Verapamil and nifedipine had no effect at concentrations that inhibit L-type Ca(2+) channels, but diltiazem inhibited SOCE by approximately 40% (≥0.1 μm). The RT-PCR demonstrated transcript expression for ORAI 1, 2, and 3, and TRPC1, 3, 4, and 7. Transcripts for TRPV1 and 2, which are activated by 2APB, were also expressed. The pharmacological profile of SOCE in retinal arteriolar smooth muscle appears unique when compared with other vascular tissues. This suggests that the molecular mechanisms underlying SOCE can differ, even in closely related tissues. Taken together, the pharmacological and molecular data are most consistent with involvement of TRPC1 in SOCE, although involvement of ORAI or other TRPC channels cannot be excluded.

Multinephron dynamics on the renal vascular network

Tubuloglomerular feedback (TGF) and the myogenic mechanism combine in each nephron to regulate blood flow and glomerular filtration rate. Both mechanisms are nonlinear, generate self-sustained oscillations, and interact as their signals converge on arteriolar smooth muscle, forming a regulatory ensemble. Ensembles may synchronize. Smooth muscle cells in the ensemble depolarize periodically, generating electrical signals that propagate along the vascular network. We developed a mathematical model of a nephron-vascular network, with 16 versions of a single nephron model containing representations of both mechanisms in the regulatory ensemble, to examine the effects of network structure on nephron synchronization. Symmetry, as a property of a network, facilitates synchronization. Nephrons received blood from a symmetric electrically conductive vascular tree. Symmetry was created by using identical nephron models at each of the 16 sites and symmetry breaking by varying nephron length. The symmetric model achieved synchronization of all elements in the network. As little as 1% variation in nephron length caused extensive desynchronization, although synchronization was maintained in small nephron clusters. In-phase synchronization predominated among nephrons separated by one or three vascular nodes and antiphase synchronization for five or seven nodes of separation. Nephron dynamics were irregular and contained low-frequency fluctuations. Results are consistent with simultaneous blood flow measurements in multiple nephrons. An interaction between electrical signals propagated through the network to cause synchronization; variation in vascular pressure at vessel bifurcations was a principal cause of desynchronization. The results suggest that the vasculature supplies blood to nephrons but also engages in robust information transfer.

Abstract 38: Rbp-j Regulates the Genetic Program of the Myo-epithelioid JG Cell

RBP-J , the major downstream effector of Notch signaling, is necessary to maintain the number of juxtaglomerular (JG) cells. In addition, RBP-J regulates the plasticity of arteriolar smooth muscle cells to adopt the renin cell phenotype when homeostasis is threatened. We hypothesized that RBP-J acts as an on/off switch controlling the expression of genes that determine the renin phenotype. To determine whether RBP-J directly affects renin gene expression, we generated mice harboring a bacterial artificial chromosome (BAC) transgene with green fluorescent protein (GFP) under the control of the renin gene carrying a mutation in its RBP-J- binding site (Mut-BAC). Mut-BAC mice had markedly reduced GFP expression to 12.9 % ±0.01 (n=3) of the control (Wt-BAC) and a diminished response to homeostatic challenges: mut-BAC mice had a reduced number of GFP positive JG areas per total number of glomeruli (Wt-BAC: 25.1 % ±3.0, n=3; Mut-BAC: 9.3 % ±1.4, n=2, p<0.02) and no GFP expression along the arterioles. To determine whether the decrease in the number of JG cells in mice lacking RBP-J (cKO) was due to a diminished endowment of renin progenitor cells, we traced the fate of cells derived from the renin lineage by generating mice ( RBP-J fl/fl ; Ren1d +/cre ; R26R +/- ) in which cells lacking RBP-J simultaneously expressed β-galactosidase (β-gal). The pattern of β-gal in cKO and control kidneys was identical, indicating that cells derived from the renin lineage did not die but instead changed their phenotype. Next we investigated the phenotype adopted by the cells derived from the renin lineage. Expression of α-smooth muscle actin and smoothelin (a marker of mature smooth muscle) was significantly decreased to 41 % ±7.0 (n=2) and 44 % ±8.8 (n=2) respectively with respect to controls (p<0.01). In addition, mutant JG cells in vivo did not express genes characteristic of the renin phenotype such as renin, calponin1, Nfat and Akr1b7 expressing instead fibroblast-specific protein 1 indicating the adoption of a fibroblast-like phenotype. Results indicate that RBP-J directly governs a genetic program that controls the dual endocrine-contractile phenotype of the JG cell, which is crucial to maintain blood pressure and fluid-electrolyte homeostasis.

P6 Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilation

Hydrogen sulfide (H 2 S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H 2 S induce vasodilation are unclear. We tested the hypothesis that H 2 S dilates piglet cerebral arterioles by modulating local and global intracellular Ca 2+ signals in smooth muscle cells. High-speed confocal imaging revealed that H 2 S increased Ca 2+ spark frequency ∼1.43-fold and decreased global intracellular Ca 2+ concentration ([Ca 2+ ] i ) by ∼37 nM in smooth muscle cells of intact cerebral arteries. In contrast, H 2 S did not alter Ca 2+ wave frequency. In voltage-clamped (−40 mV) cells, H 2 S increased the frequency of Ca 2+ spark-induced transient Ca 2+ -activated K + (K Ca ) currents ∼ 1.8-fold, but did not alter the amplitude of these events. H 2 S did not alter the activity of single K Ca channels recorded in the absence of Ca 2+ sparks in arteriole smooth muscle cells. H 2 S increased SR Ca 2+ load ([Ca 2+ ] SR ), measured as caffeine (10 mM)-induced [Ca 2+ ] i transients, ∼1.5-fold. H 2 S dilated pressurized cerebral arterioles. Iberiotoxin, a K Ca channel blocker, and ryanodine, a ryanodine receptor (RyR) channel inhibitor, reduced H 2 S-induced vasodilation by ∼38 and ∼37%, respectively. In summary, our data indicate that H 2 S elevates [Ca 2+ ] SR , leading to Ca 2+ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient K Ca current frequency leads to a reduction in global [Ca 2+ ] i and vasodilation.

Immunofluorescent visualisation of focal adhesion kinase in human skeletal muscle and its associated microvasculature

Within animal skeletal muscle, focal adhesion kinase (FAK) has been associated with load-dependent molecular and metabolic adaptation including the regulation of insulin sensitivity. This study aimed to generate the first visual images of the localisation of FAK within human skeletal muscle fibres and its associated microvasculature using widefield and confocal immunofluorescence microscopy. Percutaneous muscle biopsies, taken from five lean, active males, were frozen and 5-μm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and the microvasculature. Anti-myosin heavy chain type I was used for fibre-type differentiation. Muscle sections were also incubated with anti-dihydropyridine receptor (DHPR) to investigate co-localisation of FAK with the t-tubules. FITC-conjugated Ulex europaeus Agglutinin I stained the endothelium of the capillaries, whilst anti-smooth muscle actin stained the vascular smooth muscle of arterioles. Fibre-type differences in the intensity of FAK immunofluorescence were determined with image analysis software. In transversely and longitudinally orientated fibres, FAK was localised at the sarcolemmal regions. In longitudinally orientated fibres, FAK staining also showed uniform striations across the fibre and co-staining with DHPR suggests FAK associates with the t-tubules. There was no fibre-type difference in sarcoplasmic FAK content. Within the capillary endothelium and arteriolar smooth muscle, FAK was distributed heterogeneously as clusters. This is the first study to visualise FAK in human skeletal muscle microvasculature and within the (sub)sarcolemmal and t-tubule regions using immunofluorescence microscopy. This technique will be an important tool for investigating the role of FAK in the intracellular signalling of human skeletal muscle and the endothelium of its associated microvasculature.

Arteriolar vascular smooth muscle cells: Mechanotransducers in a complex environment

Contraction of small artery (diameters typically less than 250μm) vascular smooth muscle cells (VSMCs) plays a critical role in local control of blood flow and arterial pressure through its affect on vascular caliber. Specifically, contraction of small arteries in response to increased intraluminal pressure is referred to as the myogenic response and represents an important role for mechanotransduction. Critical questions remain as to how changes in pressure are sensed by VSMCs and transduced across the cell membrane to tune the contractile state of the cell. Recent studies suggest a pivotal role for interactions between VSMCs and extracellular matrix (ECM) proteins. Thus, pressure-induced deformation of ECM proteins and their cell surface receptors (for example, integrins) may initiate contraction and cytoskeletal remodeling through modulation of ion channels, membrane depolarization, increased intracellular Ca2+ and actomyosin crossbridge cycling. Importantly, it is argued that the contractile properties of small artery VSMCs reflect an intimate and integrated interaction with their extracellular environment and the three-dimensional structure of the vessel wall.

Hydrogen sulfide activates Ca2+ sparks to induce cerebral arteriole dilatation

Hydrogen sulfide (H₂S) is a gaseous vasodilator produced by endothelial cells. Mechanisms by which H₂S induces vasodilatation are unclear. We tested the hypothesis that H₂S dilates cerebral arterioles by modulating local and global intracellular Ca²⁺ signals in smooth muscle cells. High-speed confocal imaging revealed that Na₂S, an H₂S donor, increased Ca²⁺ spark frequency ∼1.43-fold and decreased global intracellular Ca²⁺ concentration ([Ca²⁺]i) by ∼37 nM in smooth muscle cells of intact piglet cerebral arterioles. In contrast, H₂S did not alter Ca²⁺ wave frequency. In voltage-clamped (-40 mV) cells, H₂S increased the frequency of iberiotoxin-sensitive, Ca²⁺ spark-induced transient Ca²⁺-activated K⁺ (KCa) currents ∼1.83-fold, but did not alter the amplitude of these events. H₂S did not alter the activity of single KCa channels recorded in the absence of Ca²⁺ sparks in arteriole smooth muscle cells. H₂S increased SR Ca²⁺ load ([Ca²⁺]SR), measured as caffeine (10 and 20mM)-induced [Ca²⁺]i transients, ∼1.5-fold. H₂S hyperpolarized (by ∼18 mV) and dilated pressurized (40 mmHg) cerebral arterioles. Iberiotoxin, a KCa channel blocker, reduced H₂S-induced hyperpolarization by ∼51%. Iberiotoxin and ryanodine, a ryanodine receptor channel inhibitor, reduced H₂S-induced vasodilatation by ∼38 and ∼37%, respectively. In summary, our data indicate that H₂S elevates [Ca²⁺]SR, leading to Ca²⁺ spark activation in cerebral arteriole smooth muscle cells. The subsequent elevation in transient KCa current frequency leads to membrane hyperpolarization, a reduction in global [Ca²⁺]i and vasodilatation.

TRPV1 in GABAergic Interneurons Mediates Neuropathic Mechanical Allodynia and Disinhibition of the Nociceptive Circuitry in the Spinal Cord

Neuropathic pain and allodynia may arise from sensitization of central circuits. We report a mechanism of disinhibition-based central sensitization resulting from long-term depression (LTD) of GABAergic interneurons as a consequence of TRPV1 activation in the spinal cord. Intrathecal administration of TRPV1 agonists led to mechanical allodynia that was not dependent on peripheral TRPV1 neurons. TRPV1 was functionally expressed in GABAergic spinal interneurons and activation of spinal TRPV1 resulted in LTD of excitatory inputs and a reduction of inhibitory signaling to spinothalamic tract (STT) projection neurons. Mechanical hypersensitivity after peripheral nerve injury was attenuated in TRPV1(-/-) mice but not in mice lacking TRPV1-expressing peripheral neurons. Mechanical pain was reversed by a spinally applied TRPV1 antagonist while avoiding the hyperthermic side effect of systemic treatment. Our results demonstrate that spinal TRPV1 plays a critical role as a synaptic regulator and suggest the utility of central nervous system-specific TRPV1 antagonists for treating neuropathic pain.

Histone acetyl transferases CBP and p300 are necessary for maintenance of renin cell identity and transformation of smooth muscle cells to the renin phenotype.

In response to a homeostatic threat circulating renin increases by increasing the number of cells expressing renin by dedifferentiation and re-expression of renin in arteriolar smooth muscle cells (aSMCs) that descended from cells that expressed renin in early life. However, the mechanisms that govern the maintenance and reacquisition of the renin phenotype are not well understood. The cAMP pathway is important for renin synthesis and release: the transcriptional effects are mediated by binding of cAMP responsive element binding protein with its co-activators, CBP and p300, to the cAMP response element in the renin promoter. We have shown previously that mice with conditional deletion of CBP and p300 (cKO) in renin cells had severely reduced renin expression in adult life. In this study we investigated when the loss of renin-expressing cells in the cKO occurred and found that the loss of renin expression becomes evident after differentiation of the kidney is completed during postnatal life. To determine whether CBP/p300 is necessary for re-expression of renin we subjected cKO mice to low sodium diet + captopril to induce retransformation of aSMCs to the renin phenotype. The cKO mice did not increase circulating renin, their renin mRNA and protein expression were greatly diminished compared with controls, and only a few aSMCs re-expressed renin. These studies underline the crucial importance of the CREB/CBP/p300 complex for the ability of renin cells to retain their cellular memory and regain renin expression, a fundamental survival mechanism, in response to a threat to homeostasis.

Autoregulation and conduction of vasomotor responses in a mathematical model of the rat afferent arteriole

We have formulated a mathematical model for the rat afferent arteriole (AA). Our model consists of a series of arteriolar smooth muscle cells and endothelial cells, each of which represents ion transport, cell membrane potential, and gap junction coupling. Cellular contraction and wall mechanics are also represented for the smooth muscle cells. Blood flow through the AA lumen is described by Poiseuille flow. The AA model's representation of the myogenic response is based on the hypothesis that changes in hydrostatic pressure induce changes in the activity of nonselective cation channels. The resulting changes in membrane potential then affect calcium influx through changes in the activity of the voltage-gated calcium channels, so that vessel diameter decreases with increasing pressure values. With this configuration, the model AA maintains roughly stable renal blood flow within a physiologic range of blood flow pressure. Model simulation of vasoconstriction initiated from local stimulation also agrees well with findings in the experimental literature, notably those of Steinhausen et al. (Steinhausen M, Endlich K, Nobiling R, Rarekh N, Schütt F. J Physiol 505: 493-501, 1997), which indicated that conduction of vasoconstrictive response decays more rapidly in the upstream flow direction than downstream. The model can be incorporated into models of integrated renal hemodynamic regulation.

A Functional Role for N‐cadherin in Mediating Mechanotransduction in Arteriolar Vascular Smooth Muscle Cells

A hybrid atomic force (AFM)‐confocal microscopy system was used to examine whether mechanical forces applied through N‐cadherin adhesions on vascular smooth muscle cells (VSMC) could induce i) cellular contraction and ii) signaling events. AFM probes were modified by attaching a borosilicate microsphere (5 μm) to the tip and coating with either 1) a recombinant N‐cadherin chimera fusion protein (rhNcad), or 2) an N‐cadherin antibody; or 3) attaching a live VSMC to the end of the AFM cantilever. In all 3 cases, a strong adhesion was formed between the AFM probe and cell membrane; and AFM pulling forces (~1000 pN) induced a strong VSMC contractile response that pulled the probe oppositely. In addition, clustering of N‐cadherin‐GFP and RhoA‐GFP was observed when an rhNcad coated AFM probe was engaged on the VSMC membrane, and application of AFM pulling force enhanced the clustering. Immuno‐fluorescence labeling showed a dramatic increase of N‐cadherin clustering in the vessel wall of pressurized arterioles that developed spontaneous myogenic tone compared to unpressurized vessels, as well as spatial correlations between the N‐cadherin and the clusters of active RhoA, myosin II and the phosphorylated myosin phosphatase targeting subunit. Collectively, these results support an important role for N‐cadherin in mediating the myogenic mechanotransduction in VSMC. (AHA 0835676N to Z.S. and NIH P01HL095486 to G.A.M.)