Modulation Of Smooth Muscle Tone Research Articles

Activation of endothelial IKCa channels underlies NO-dependent myoendothelial feedback

Agonist-induced vasoconstriction triggers a negative feedback response whereby movement of charged ions through gap junctions and/or release of endothelium-derived (NO) limit further reductions in diameter, a mechanism termed myoendothelial feedback. Recent studies indicate that electrical myoendothelial feedback can be accounted for by flux of inositol trisphosphate (IP3) through myoendothelial gap junctions resulting in localized increases in endothelial Ca2+ to activate intermediate conductance calcium-activated potassium (IKCa) channels, the resultant hyperpolarization then conducting back to the smooth muscle to attenuate agonist-induced depolarization and tone. In the present study we tested the hypothesis that activation of IKCa channels underlies NO-mediated myoendothelial feedback. Functional experiments showed that block of IP3 receptors, IKCa channels, gap junctions and transient receptor potential canonical type-3 (TRPC3) channels caused endothelium-dependent potentiation of agonist-induced increase in tone which was not additive with that caused by inhibition of NO synthase supporting a role for these proteins in NO-mediated myoendothelial feedback. Localized densities of IKCa and TRPC3 channels occurred at the internal elastic lamina/endothelial-smooth muscle interface in rat basilar arteries, potential communication sites between the two cell layers. Smooth muscle depolarization to contractile agonists was accompanied by IKCa channel-mediated endothelial hyperpolarization providing the first demonstration of IKCa channel-mediated hyperpolarization of the endothelium in response to contractile agonists. Inhibition of IKCa channels, gap junctions, TRPC3 channels or NO synthase potentiated smooth muscle depolarization to agonists in a non-additive manner. Together these data indicate that rather being distinct pathways for the modulation of smooth muscle tone, NO and endothelial IKCa channels are involved in an integrated mechanism for the regulation of agonist-induced vasoconstriction.

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Characterization of receptors involved in serotonin contractions of isolated human umbilical artery in uncomplicated pregnancy

Background Serotonin (5-HT), is a vasoactive substance involved in physiological functioning of fetoplacental blood flow and continuous modulation of smooth muscle tone in umbilical vein and arteries. Since umbilical blood vessels have been shown to be deficient in autonomic innervation, the action of local autocrine vasoactive substances (including serotonin) is of considerable importance. Although seven main groups of 5-HT receptors (5-HT1-7) have been characterized, it has been established that only several 5-HT receptor subtypes may be involved in vascular effects of this biogenic amine. Therefore, the aim of this study was to characterize receptors involved in serotonin-induced effect on isolated human umbilical artery (HUA) in uncomplicated pregnancy.

Purinergic agonists flex vas deferens muscle

The vas deferens is described typically as a muscular tube that functions as a conduit to move sperm from the epididymis to the urethra. A growing body of evidence, however, shows that the system is far more complex. Smooth muscle tone and contractions are affected by a number of neuocrine, endocrine and perhaps lumicrine agents. Likewise, epithelial activity is modified by a similar host of agents. Stimulation of the hypogastric nerve, which innervates the male reproductive ducts, results in the co-release of noradrenaline, ATP, and other neurotransmitters that induce or modulate smooth muscle contraction and can induce epithelial ion transport. In fact, it has been suggested that purinergic receptors in the reproductive duct might be targeted for the development of non-steroidal male contraceptives (Gur et al. 2007). Thus, there is great interest in determining regulatory cascades that link purinergic stimulation to smooth muscle contraction or relaxation. One caveat in exploiting purinergic pathways to manage male fertility is the apparently conflicting observations that ATP exposure can, depending upon conditions, lead to either smooth muscle contraction or relaxation. Thus, multiple receptors and signalling cascades are probably involved in these responses. Both ionotropic P2X and metabotropic P2Y receptors are present on vas deferens smooth muscle cells with rapid muscle contraction typically associated with P2X-mediated Ca2+ entry. The cascade accounting for relaxation appears to be more complex. In a recent issue of The Journal of Physiology, Ruan et al. (2008) provide keen insight on a mechanism that probably contributes to muscle relaxation, in vitro. Their work builds on the observation that vas deferens epithelium contributes to the response of smooth muscle to adrenergic stimuli (Okpalaugo et al. 2002). This observation suggests that vas deferens smooth muscle activity can be modified by paracrine agents released from the epithelium. It is becoming more widely recognized that neurotransmitters and hormones can modify the activity of epithelial cells lining the epididymes and vas deferens. Indeed, neurotransmitters, steroid hormones, peptide hormones and autocoids have been shown to modify epithelial ion transport. HCO3− secretion is particularly important because it initiates and supports increased sperm motility. Importantly, noradrenaline, ATP and adenosine have been shown to induce anion secretion. The Ruan paper adds purinergic modulation of smooth muscle tone to the activities probably mediated by the epithelium lining the reproductive duct. Prostaglandin E2 is a key element in the epithelium-dependent response to ATP. This observation has far-reaching impact because prostaglandin H synthase 2 (PGHS2; formerly known as COX2) expression in vas deferens epithelial cells is dependent upon testosterone. In both rats (McKanna et al. 1998) and humans (Kirschenbaum et al. 2000), vas deferens epithelial PGHS2 expression correlates with serum testosterone. PGHS2 immunoreactivity is present during gestation and following puberty, but absent pre-pubertally or following castration. Recently, Pierucci-Alves & Schultz (2007) demonstrated that testosterone exposure enhances bradykinin-induced vas deferens epithelial anion secretion in vitro, a response mediated by prostaglandins. Thus, testosterone-induced epithelial PGHS2 expression provides the basis for androgen-dependent sperm development and delivery. The results provided by Ruan and colleagues are the foundation of a model that brings many seemingly disparate elements together with the culmination of viable sperm delivery. ATP released from the hypogastric nerve can rapidly promote smooth muscle contraction by the activation of P2X receptors while co-released noradrenaline can promote a sustained response by activating α1 receptors on smooth muscles. These same neurotransmitters can affect epithelial HCO3− secretion by interacting with epithelial P2Y and β2 receptors, respectively. In epithelial cells exposed to testosterone, PGHS2 can be stimulated downstream from the P2Y receptor resulting in PGE2 production. PGE2 then interacts with sperm to promote motility, with prostanoid receptors on epithelial cells to further enhance anion secretion, and with prostanoid receptors on adjacent smooth muscle cells to promote hyperpolarization and smooth muscle relaxation. Thus, the work presented in this issue provides compelling evidence that the vas deferens is much more that just a muscular tube. The vas deferens is a complex tissue in which many component parts contribute to the timely delivery of mature active sperm. Dr Schultz receives research support from the National Institutes of Health (R01HD058398).

Contribution of potassium channels in human umbilical artery contractions

Bradykinin (BK) and serotonin (5-HT) are vasoactive agents that significantly contribute to the normal functioning of fetoplacental blood flow, including modulation of smooth muscle tone in umbilical vein and arteries. Accordingly, umbilical artery is a crucial component of fetoplacental circulation with the vital function of providing suitable perfusion and nutrition for placenta. The aim of this study was to investigate the effect of potassium channel blockers, namely glibenclamide (an ATP-sensitive K+ channel blocker), tetraethylammonium (a non-selective K+ channel blocker), Ba2+ (an inwardly rectifying K+ channel blocker) and apamin (a small conductance Ca2+-activated K+ channel blocker) in BK- and 5-HT-induced actions on isolated human umbilical artery (HUA) in normal pregnancy. The experiments were performed on vascular rings of HUA isolated from umbilical cords that were obtained immediately after vaginal delivery in women with uncomplicated pregnancy. Only the remnant tissue, which would have been otherwise disposed of, has been utilized. Isometric tension of suspended artery rings was continuously recorded. BK (1 nM – 1 μM) and 5-HT (1 nM – 30 μM) produced concentration-dependent contractions of HUA. Control contractions produced by BK were notably (p < 0.05) enhanced only in the presence of apamin (20 μM), while unaltered by other inhibitors. 5-HT-induced effect was not influenced by any of applied blockers. Our results suggest that small conductance Ca2+-activated potassium channels contribute to the fine regulation of maximal BK-induced contraction of HUA in normal pregnancy. On the other hand, potassium channels do not seem to be involved in 5-HT-evoked contractile response of umbilical artery.

Nitric oxide modulation of ophthalmic artery blood flow velocity waveform morphology in healthy volunteers

Quantitative analysis of the arterial pressure pulse waveform recorded by applanation tonometry of the radial artery can track NO (nitric oxide)-mediated modulation of arterial smooth muscle tone. The changes in pressure pulse waveform morphology result from pulse wave reflection arising predominantly from smaller arteries and arterioles. Employing Doppler ultrasound to record the spectral flow velocity waveform in the ophthalmic artery, we studied the effects of NO modulation on waveforms recorded in the proximity of the terminal ocular microcirculatory bed. In healthy young men (n=10; age 18-26 years), recordings were made at baseline, following 300 mug of sublingual GTN (glyceryl trinitrate) and during the intravenous infusion of 0.25 and 0.5 mg/kg of L-NAME (N(G)-nitro-L-arginine methyl ester). Peaks (P1, P2 and P3) and nodes (N1, N2 and N3) on the arterial flow velocity waveform were identified during the cardiac cycle and employed to quantify wave shape change in response to the haemodynamic actions of the pharmacological interventions. The administration of GTN resulted in a significant (P<0.05) increase in heart rate without significant alteration in blood pressure. At the doses employed, L-NAME did not significantly alter systemic haemodynamics. With the exception of peak Doppler systolic velocity, all other peaks and nodes decreased significantly in response to GTN (P<0.05 for all points compared with baseline). In response to the administration of L-NAME, all peaks and nodes decreased significantly (P<0.05 for all points compared with baseline). The resistive index, a ratio calculated from the peak and trough flow velocities employed to assess change in flow resistance, increased significantly in response to GTN (0.77 at baseline compared with 0.85; P<0.05). Quantification of changes in the flow velocity spectral waveform during the cardiac cycle sensitively identified NO modulation of smooth muscle tone prior to alteration in systemic haemodynamics. Focusing on the resistive index, which identifies isolated points on the waveform describing the excursions of flow, may provide misleading information in relation to the haemodynamic effects of drug interventions.

Functional role of TRPC proteins in native systems: implications from knockout and knock‐down studies

Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease.

Physiological roles for K+ channels and gap junctions in urogenital smooth muscle: implications for improved understanding of urogenital function, disease and therapy.

Smooth muscle cells constitute a heterogeneous collection of effector cells that, by virtue of both their constituency in blood vessels and presence as primary parenchymal cells in diverse tissues, affect the function of all organs. Thus, perhaps it is not surprising that alterations in, and/or dysfunction of, smooth muscle cells are quite common, and responsible, at least in part, for the morbidity and mortality associated with a very wide range of human diseases. These facts point to the necessity for improved understanding of the mechanism(s) governing the control of myocyte contractility (i.e., tone). Such understanding has been rapidly forthcoming in recent years, and has indicated that in many smooth muscle cell types intercellular communication through gap junctions acts in concert with nonjunctional (K+) ion channels to make important contributions to the control of myocyte tone and tissue homeostasis in physiologically diverse organs. Intercellular communication through connexin43-derived gap junction channels and K+ flux through the KCa and KATP channel subtypes, in particular, appear to play prominent roles in this process. The goal of this report, therefore, is to review the data concerning junctional and nonjunctional ion channels on the detrusor myocytes of the urinary bladder, as well as on the specialized vascular myocytes of the corpus cavernosum. The choice of an excitable (i.e., bladder detrusor myocytes) and nonexcitable (i.e., corporal smooth muscle) smooth muscle cell type ensures that the discussion will at least encompass consideration of a large portion of the spectrum of physiological possibilities for the participation of junctional and nonjunctional ion channels in the initiation, maintenance and modulation of smooth muscle tone. A central thesis of this communication is that detailed knowledge of the myocyte- and tissue-specific properties of K+ channels and gap junctions will likely lead to the improved understanding and treatment of human smooth muscle diseases/disorders.

Multiple types of ion channels in cavernous smooth muscle.

The excitation process of the smooth muscle of corpus cavernosum is important to the erectile process. single-cell electrophysiology performed during the last decade has given deep insights into the different current components participating in that process. However, the existence of a tremendous amount of literature might be confusing for the nonspecialist in the field of smooth-muscle excitation and, especially, in the excitation of corpus cavernosum. In a compact form, this paper gives an overview on significant ionic currents as well as their ability to change the membrane potential and to enhance or inhibit calcium influx for the modulation of smooth-muscle tone.

Endothelin in the Urinary Bladder. II. Characterization of Endothelin Receptor Subtypes

Endothelins (ET-1, ET-2, ET-3) are a family of regulatory peptides with diverse biological functions, including modulation of smooth muscle tone. To evaluate the possible role of endothelins in the control of the detrusor smooth muscle of urinary bladder, we have investigated the responses of isolated strips of bladder dome and base to endothelins, in organ chambers, and characterized the receptors for these isopeptides in bladder membranes. ET-1, ET-2 and to a lesser extent ET-3 caused sustained concentration-dependent contraction of bladder dome and base. The contraction to endothelins, unlike the short-lived contraction produced by cholinergic agents, was long lasting and difficult to wash out. Equilibrium binding studies demonstrated that endothelin isoforms bound to bladder membrane receptors specifically, with high affinity (KD of 0.4–0.6nM) and limited capacity (60-420 fmol/mg protein). Competition analysis showed two populations of receptors: one with high affinity for ET-1 and ET-2 and low affinity for ET-3 and another with high affinity for ET-1, ET-2 and ET-3. Chemical affinity labeling of endothelins to bladder membranes demonstrated that ET-1 and ET-2 were cross-linked to three proteins (75, 52 and 34kDa, respectively), whereas ET-3 was cross-linked mainly to a 34kDa protein. The data obtained from equilibrium binding studies, competition analysis and cross-linking experiments suggest that at least two endothelin receptor subtypes exist in bladder tissue. These observations further suggest that bladder smooth muscle tone may be modulated by endothelin or an endothelin-like substance via interaction with specific receptor sites.