Smooth Muscle Membrane Research Articles

Nitric Oxide Suppresses Cerebral Vasomotion by sGC-Independent Effects on Ryanodine Receptors and Voltage-Gated Calcium Channels

Background/Aims: In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. Methods: In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca²⁺] ([Ca²⁺]_SMC) changes were recorded. Results: In the absence of L-NAME, asynchronous propagating Ca²⁺ waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L-NAME stimulated pronounced vasomotion and synchronous Ca²⁺ oscillations with close temporal coupling between membrane potential, tone and [Ca²⁺]_SMC. If nifedipine was applied together with L-NAME, [Ca²⁺]_SMC decreased and synchronous Ca²⁺ oscillations were lost, but asynchronous propagating Ca²⁺ waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L-NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK_Ca channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca²⁺ channels (VGCC), which was independent of both voltage and sGC. Conclusion: NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone.

Mutation of protein kinase C phosphorylation site S1076 on α-subunits affects BKCachannel activity in HEK-293 cells

Large conductance, calcium- and voltage-activated potassium (BK(Ca)) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK(Ca) channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK(Ca) channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK(Ca) channel alpha-subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK(Ca) channels. Results showed that mutating S1076 altered the effect of PKC activation on BK(Ca) channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK(Ca)-alpha(S1076A)/beta(1) attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK(Ca) channels, whereas the phospho-mimetic mutation BK(Ca)-alpha(S1076E)/beta(1) increased the excitatory effect of PMA on BK(Ca) channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK(Ca) channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK(Ca) channel alpha-subunits influences BK(Ca) channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

ABNORMAL ACTIVATION OF POTASSIUM CHANNELS IN AORTIC SMOOTH MUSCLE OF RATS WITH PERITONITIS-INDUCED SEPTIC SHOCK

This study was conducted to examine the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by cecal ligation and puncture (CLP) in endothelial-denuded strips of rat thoracic aorta ex vivo. The CLP for 18 h elicited a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine as seen in severe sepsis. At the end of the in vivo experiments, thoracic aortas were removed from both CLP-treated and control rats. After removal of the endothelium, aortic segments were mounted in myographs for the recording of isometric tension and smooth muscle membrane potential. The membrane potential recording showed that a hyperpolarization was observed in the CLP-treated rats when compared with the control rats. This hyperpolarization was reversed by iberiotoxin (a large-conductance Ca2+-activated K+ channel blocker), 4-aminopyridine (a voltage-dependent K+ channel blocker), barium (an inward rectifier K+ channels blocker), N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (a pore-forming blocker of adenosine triphosphate (ATP)-sensitive K+ channels [KATP]), or methylene blue (a nonspecific guanylyl cyclase [GC] inhibitor). However, this hyperpolarization was not significantly affected by apamin (a small-conductance Ca2+-activated K+ channel blocker), glibenclamide (a sulfonylurea blocker of KATP), N(omega)-nitro-L-arginine methyl ester (a NOS inhibitor), or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an NO-sensitive GC inhibitor). In addition, the basal tension of the tissues obtained from CLP rats was increased simultaneously, whereas membrane potential was reversed. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Thus, we provide electrophysiological and functional evidence demonstrating that an abnormal activation of K+ channels in vascular smooth muscle in animals with septic shock induced by CLP. Our observations suggest that the activation of large conductance Ca2+-activated K+ channels, voltage-dependent K+ channels, inward rectifier K+ channels, and KATP channels, but not small conductance Ca2+-activated K+ channels, contributes to CLP-induced vascular hyporeactivity. Furthermore, the hyperpolarization in septic shock induced by CLP is likely via non-NO-sensitive GC pathway.

A role for nitroxyl (HNO) as an endothelium‐derived relaxing and hyperpolarizing factor in resistance arteries

Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium-dependent relaxation and hyperpolarization in resistance arteries. Rat and mouse mesenteric arteries were mounted in small vessel myographs for isometric force and smooth muscle membrane potential recording. Vasorelaxation to the HNO donor, Angeli's salt, was attenuated in both species by the soluble guanylate cyclase inhibitor (ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one), the voltage-dependent K(+) channel inhibitor, 4-aminopyridine (4-AP) and the HNO scavenger, L-cysteine. In mouse mesenteric arteries, nitric oxide (NO) synthase inhibition (with L-NAME, N(omega)-Nitro-L-arginine methyl ester) markedly attenuated acetylcholine (ACh)-mediated relaxation. Scavenging the uncharged form of NO (NO(*)) with hydroxocobalamin (HXC) or HNO with L-cysteine, or 4-AP decreased the sensitivity to ACh, and a combination of HXC and L-cysteine reduced ACh-mediated relaxation, as did L-NAME alone. ACh-induced hyperpolarizations were significantly attenuated by 4-AP alone and in combination with L-NAME. In rat mesenteric arteries, blocking the effects of endothelium-derived hyperpolarizing factor (EDHF) (charybdotoxin and apamin) decreased ACh-mediated relaxation 10-fold and unmasked a NO-dependent component, mediated equally by HNO and NO(*), as HXC and L-cysteine in combination now abolished vasorelaxation to ACh. Furthermore, ACh-evoked hyperpolarizations, resistant to EDHF inhibition, were virtually abolished by 4-AP. The factors contributing to vasorelaxation in mouse and rat mesenteric arteries are NO(*) = HNO > EDHF and EDHF > HNO = NO(*) respectively. This study identified HNO as an endothelium-derived relaxing and hyperpolarizing factor in resistance vessels.

Endothelial dysfunction and vascular disease

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor (EDRF) is nitric oxide (NO). The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium-dependent hyperpolarizations, EDHF-mediated responses). Endothelium-dependent relaxations involve both pertussis toxin-sensitive G(i) (e.g. responses to serotonin and thrombin) and pertussis toxin-insensitive G(q) (e.g. adenosine diphosphate and bradykinin) coupling proteins. The release of NO by the endothelial cell can be up-regulated (e.g. by oestrogens, exercise and dietary factors) and down-regulated (e.g. oxidative stress, smoking and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively loose the pertussis toxin-sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and causing endothelium-dependent hyperpolarizations), endothelial cells also can evoke contraction (constriction) of the underlying vascular smooth muscle cells by releasing endothelium-derived contracting factor (EDCF). Most endothelium-dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells. EDCF-mediated responses are exacerbated when the production of NO is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium-dependent vasodilatations in aged subjects and essential hypertensive patients.

Sustainable TRPM4 Channel Activity in Freshly‐Isolated Cerebral Artery Smooth Muscle Cells

Localized control of blood flow and arterial constriction in response to changes in intraluminal pressure are dependent on the intrinsic regulation of smooth muscle cell membrane potential. The melastatin transient receptor potential (TRP) channel TRPM4 is highly selective for monovalent cations, is activated by elevated levels of intracellular Ca2+, and is a critical regulator of smooth muscle membrane depolarization, which serves to open voltage dependent Ca2+channels (VDCC), and resulting in myocyte contraction. Interestingly, Ca2+ activates and inactivates TRPM4, causing the channel to rapidly inactivate under conventional whole‐cell patch clamp conditions. When the slow Ca2+ buffer, ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA), was included in the pipette solution, we identified a transient tetraethylammonium (TEA)‐insensitive inward Na+ current in freshly isolated cerebral arterial myocytes. siRNA‐mediated silencing of TRPM4 and the recently described TRPM4 inhibitor, 9‐phenanthrol, both diminish the open probability (NPo) compared to controls, strongly suggesting the molecular identity of the channel to be TRPM4. This study demonstrates a practical method to record sustained TRPM4 activity and allows for further investigation into mechanisms of channel regulation. AHA0535226N; F31HL094145‐01.

Participation of Rho-associated kinase in electrical stimulated and acetylcholine-induced contraction of feline esophageal smooth muscle

The RhoA/Rho-associated kinase (ROCK) signaling pathway has been known to play a critical role in Ca 2+-sensitization of smooth muscle contraction. In this study, we investigated the role of ROCK in feline esophageal body smooth muscle contraction induced by electrical field stimulation and exogenous acetylcholine in vitro. Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl) cyclohexanecarboxamide dihydrochloride], ROCK inhibitor, and specific antibodies to ROCK1 and ROCK2 proteins, which are two isoforms of ROCK, were used. Electrical field stimulation induced off-contraction and on-contraction in the presence of N G-nitro-L-arginine methylester, originating from the cholinergic nerve. Y-27632 inhibited both excitatory contractions in a concentration-dependent manner. Exogenous acetylcholine concentration-dependently induced two types of contractions: an initial contraction which occurred immediately after the addition of acetylcholine during short periods, and a sustained contraction which sluggishly continued after the initial contraction. Maximal initial and sustained contractions were reached at 10 − 5 M acetylcholine. Y-27632 significantly inhibited both acetylcholine-induced contractions in a concentration-dependent manner. Western blot analysis revealed that acetylcholine maximally increased the level of phosphorylation in the 20 kDa regulatory light chain of myosin II (MLC 20) at Ser 19 from 0.25 min to 1 min, and then declined after 2 min. The level changes of MLC 20 phosphorylation during the 5 min paralleled with those of acetylcholine-induced contractions. The expression of ROCK1 and ROCK2 in membrane fractions of muscle was increased by acetylcholine; more specifically, ROCK2 continually expressed up to 5 min. Taken together, ROCK may be involved in neural-evoked and acetylcholine-induced contraction via translocation to the membrane in feline esophageal smooth muscle.

TRPCs, GPCRs and the Bayliss effect

An increase in pressure inside most small arteries unexpectedly results in a vasoconstriction. This phenomenon, known as the Bayliss effect, involves the stretch‐induced activation of non‐selective cation channels in the vascular smooth muscle cells. Recent work by Mederos y Schnitzler et al . now demonstrates that the stretch‐induced channel activity originates from a fascinating interplay between a TRP cation channel and the angiotensin receptor.

Chlamydia Infection Causes Loss of Pacemaker Cells and Inhibits Oocyte Transport in the Mouse Oviduct1

Chlamydia trachomatis is a common sexually transmitted bacterial infection that results in health care costs in the United States that exceed $2 billion per year. Chlamydia infections cause damage to the oviducts, resulting in ectopic pregnancy and tubal factor infertility, but the reasons for defective oviduct function are poorly understood. We have investigated the role of oviduct contractions in egg transport and found that underlying electrical pacemaker activity is responsible for oviduct motility and egg transport. Specialized pacemaker cells, referred to as oviduct interstitial cells of Cajal (ICC-OVI), are responsible for pacemaker activity. The ICC-OVI, labeled with antibodies to KIT protein, form a dense network associated with the smooth muscle cells along the entire length of the oviduct. Selective removal of ICC-OVI with KIT-neutralizing antibody resulted in loss of electrical rhythmicity and loss of propulsive contractions of the oviduct. We tested whether infection might adversely affect the ICC-OVI. Mice infected with Chlamydia muridarum displayed dilation of oviducts, pyosalpinx, and loss of spontaneous contractile activity. Morphological inspection showed disruption of ICC-OVI networks, and electrophysiological recordings showed loss of intrinsic pacemaker activity without change in basal smooth muscle membrane potential. Chlamydia infection also was associated with upregulation of NOS2 (iNOS) and PTGS2 (COX II) in leukocytes. Loss of ICC-OVI and pacemaker activity causes oviduct pseudo-obstruction and loss of propulsive contractions for oocytes. This, accompanied by retention of oviduct secretions, may contribute to the development of tubal factor infertility.

Mechanism of ATP-Induced Local and Conducted Vasomotor Responses in Isolated Rat Cerebral Penetrating Arterioles

Background: Adenosine triphosphate (ATP), a potent vascular regulator in the cerebral circulation, initiates conducted vasomotor responses which may be impaired after pathological insults. We analyzed the mechanism of ATP-induced local vasomotor responses and their effect on conducted vasomotor responses in rat cerebral penetrating arterioles. Methods: Arterioles were cannulated and their internal diameter monitored. Vasomotor responses to ATP were observed in the presence or absence of inhibitors, or after endothelial impairment. Smooth muscle membrane potentials were measured in some vessels. Results: Microapplication of ATP produced a biphasic response (constriction followed by dilation), which resulted in conducted dilation preceded by a membrane hyperpolarization. α,β-methylene-ATP or pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blunted the ATP-mediated constriction and enhanced local and conducted dilation. N^ω-monomethyl-L-arginine, endothelial impairment and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) reduced the local dilation caused by ATP. The conducted dilation was attenuated by MS-PPOH and endothelial impairment, but not N^ω-monomethyl-L-arginine or indomethacin. Conclusion: ATP-induced conducted dilation is preceded by membrane hyperpolarization. Local ATP induces initial local constriction via smooth-muscle P_2X1 and subsequent dilation via endothelial P_2Y receptors. Nitric oxide, cytochrome P450 metabolites, and intermediate and large conductance K_Ca channels mediate dilation caused by ATP. ATP-induced conducted dilation is dependent upon both the endothelium and cytochrome P450 metabolites.

HIV-1 infection can infect and replicate in human macrophages undetected by the host innate immune system

Abstract Introduction: A key element in the management of septic shock is maintenance of a blood pressure adequate for organ perfusion. Patients in prolonged septic shock have impaired pressor responses to norepinephrine (NE) yet enhanced sensitivity to vasopressin (VP). As both cause vasoconstriction through G protein-coupled receptors, activating the inositol phosphate cascade to increase vascular smooth muscle Ca2+ levels, the reason for this disparity is uncertain. We postulated that these drugs may have diverse effects on different Ca2+ mobilisation pathways during sepsis. We investigated this hypothesis using specific modulators of Ca2+ release and influx on the contractile responses to VP and NE in mesenteric arteries taken from septic and sham-operated rats. Methods: Sepsis was induced in 6 awake, fluid-resuscitated Wistar rats by i.p. injection of faecal slurry. Paired sham controls received no injection. Rats were sacrificed after 24 hours, and mesenteric arteries mounted on a wire myograph to measure isometric tension responses to VP and NE. The contributions of sarcoplasmic reticulum Ca2+ release and Ca2+ entry through the store-operated channel (SOC) were assessed by removing and returning extracellular Ca2+ respectively. The contribution of the voltage-gated Ca2+ channel (VGCC) was assessed by applying VP/NE in the presence of nifedipine. Results: Mimicking the clinical scenario, contractions were significantly enhanced to VP but depressed to NE in septic vessels (p<0.05). In all arteries, constriction to both agonists relied predominantly on extracellular Ca2+ influx rather than Ca2+ release from sarcoplasmic reticulum. The Ca2+ influx in response to NE was almost entirely VGCC-mediated, with a negligible contribution from SOCs in both sham and septic arteries. On the other hand, SOCs contributed significantly to VP-induced contraction, and SOCrather than VGCC-mediated influx of Ca2+ predominated in septic arteries. Discussion: Despite coupling to similar 2nd messenger systems, VP and NE appear to differentially activate SOCand VGCC-mediated Ca2+ entry. In sepsis, hyperpolarisation of the vascular smooth muscle membrane may impair VGCC activity. The ability of VP to preferentially activate SOCs may explain its preserved vasoconstrictor function in septic shock. Ca2+ channel modulation in this context is a potential new therapeutic paradigm.

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A 2 is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP− and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP− rabbits. Radioligand binding studies were performed with 125I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP− rabbit aortas, no measurable binding to 125I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP− rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP− compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 μM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 μM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP− was not altered by U46619. In a separate study, U46619 (3 μM) increased the release of 6-keto PGF 1α, the stable metabolite of prostacyclin, in vTP+ but not vTP− endothelial cells. Pretreatment with SQ29548 (10 μM) or the cyclooxygenase inhibitor, indomethacin (10 μM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.

The effect of ovariectomy and oestrogen therapy on the free fatty acid content, endogenous lipase activity, and phospholipid content of the rabbit urinary bladder

To evaluate the effects of ovariectomy (Ovx) and oestrogen therapy on the free fatty acid (FFA) content, endogenous lipase activity, and the phospholipid (PL) content of the urinary bladder, as reduced circulating oestrogen during and after the menopause has been linked to various bladder dysfunctions including incontinence and recurrent urinary tract infections. In all, 12 New Zealand White female rabbits were separated into three groups; the Ovx + oestrogen group received Ovx and treatment with conjugated beta-oestradiol (3 weeks), the Ovx group received Ovx and vehicle (3 weeks), and the control group received sham operation and vehicle. Cystometry was used to evaluate compliance and in vitro muscle strip studies quantified contractility. For the bulk of the bladder, the muscle and mucosa were separated; FFA and PL concentrations were analysed using standard biochemical techniques. The bladder contractile responses and compliance decreased after Ovx and returned to or above normal after oestrogen administration. Both FFA and PL concentrations of the mucosa were about three times greater than that of the smooth muscle. Ovx significantly reduced the FFA and PL concentrations of both muscle and mucosa, while oestrogen therapy restored them to normal. Reduced FFA and PL content of the smooth muscle membranes would decrease their fluidity and contribute to decreased compliance and contractility. Reduced FFA and PL content of the mucosa would be consistent with mucosal damage and may contribute to the increased incidences of incontinence and bladder infection.

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.

Localization of CCK-1R in the omasum and role of CCK in the regulation of omasal contractions in sheep

The present study examined localization of cholecystokinin receptor (CCK-R) mRNA in the muscle layer of the ovine omasum and role of CCK-R type 1 (CCK-1R) in the regulation of muscle contraction of the omasum. We demonstrated that not only CCK-R type 2 (CCK-2R) mRNA but also CCK-1R mRNA is highly expressed in the muscle layer of the ovine omasum. Application of CCK-8 to muscle strips of the greater curvature of the ovine omasum at 1–100 nM induced tonic contraction in a concentration-dependent manner, and the contractile effect of CCK-8 was inhibited by both CCK-1R antagonist lorglumide (IC 50 2.7 and 7.9 μM in the longitudinal and circular muscle, respectively) and CCK-2R antagonist PD135,158 (IC 50 51.4 μM in the longitudinal muscle), indicating that not only CCK-2R but also CCK-1R is functionally expressed in the plasma membrane of smooth muscles in the omasum and mediates action of exogenous CCK. Contractile effect of intravenous infusion of CCK-8 (1–30 pmol/kg/min) on omasal contraction was also confirmed in the in vivo experiments using conscious sheep in the absence and presence of atropine infusion (14.4 nmol/kg/min), and showed that circulating CCK increases omasal electromyographic (EMG) activity at lower plasma concentration than that it inhibits ruminal contractions. Taking account of our previous results in the in vivo study using other CCK-1R antagonist, it is suggested that circulating CCK, even at normal range of plasma concentration, plays a physiological role as a regulator of omasal contractions in sheep and CCK-1R mediates the action of CCK.

C-Type Natriuretic Peptide Hyperpolarizes and Relaxes Human Penile Resistance Arteries

In addition to nitric oxide (NO), it is thought that an endothelium-derived hyperpolarizing factor (EDHF) plays an important role in the relaxation of penile arteries. Recently, it has been shown that C-type natriuretic peptide (CNP) shows the characteristics of EDHF in systemic small arteries. To investigate the mechanism involved in CNP-evoked vasodilatation and to address whether CNP is an EDHF in human penile resistance arteries. Erectile tissue was obtained in connection with transsexual operations. Intracavernous penile resistance arteries were isolated and mounted in microvascular myographs for recording of isometric tension. Membrane potential was recorded by the use of a small glass electrode inserted in the smooth muscle layer. In vitro evidence for hyperpolarization and vasorelaxation induced by CNP. Acetylcholine (ACh) and CNP hyperpolarized smooth muscle membrane potential in resting penile resistance arteries. In penile small arteries incubated with inhibitors of NO synthase and cyclooxygenase and contracted with phenylephrine, ACh and CNP evoked concentration-dependent relaxations with maximum of 56 +/- 6% and 71 +/- 6%, respectively. Addition of a combination of blockers of small- and intermediate-conductance calcium-activated K(+) channels, apamin plus charybdotoxin, respectively, and a combination thought to block the smooth muscle response of EDHF-type relaxation, barium plus ouabain, markedly reduced ACh- and CNP-evoked relaxation. Iberiotoxin, a blocker of big-conductance calcium-activated K(+) channels inhibited the vasorelaxant responses evoked by ACh and CNP. A selective natriuretic peptide receptor type C (NPR-C) agonist, C-atrial natriuretic factor(4-23) (cANF(4-23)), induced relaxations with less maximum response compared to CNP. The present findings suggest that CNP possesses the characteristics of an EDHF in human penile resistance arteries. By activation of natriuretic peptide receptor type B and NPR-C receptors, CNP causes relaxation by activation, respectively, of large-conductance calcium-activated K(+) channels and Na(+)/K(+)-adenosine triphosphatase (ATPase), and barium-sensitive inward rectifier K(+) channels. Modulation of the CNP pathway opens for new treatment modalities of erectile dysfunction.

Mutation of the Caveolin‐Scaffolding Domain Reduces Phosphofructokinase Localization to the Plasma Membrane in Vascular Smooth Muscle

We have previously demonstrated the role of caveolin‐1(CAV‐1) as a scaffold to localize glycolytic enzymes to the plasma membrane. Because the caveolin scaffolding domain (CSD) has been implicated in the interaction of caveolins with proteins that reside in the caveolae we sought to investigate the role of the CSD in targeting PFK in smooth muscle. We disrupted the CSD's ability to target PFK by site directed mutagenesis of a single residue without altering the membrane localization of the mutant CAV‐1. Using in vitro site directed mutagenesis we mutated aromatic amino acid phenylalanine F92, a highly conserved residue found in all three caveolins isoforms that is essential for interaction in vivo, to amino acid residue glycine. We previously reported CAV‐1 overexpression resulted in the targeting of PFK to the membrane. Here we report that only 34.3 % of PFK localized with CAV‐1 after overexpression of mutant CAV‐1, in contrast to 69.2 % of PFK which localized with CAV‐1 after overexpression of wild type CAV‐1. These results show CAV‐1 membrane distribution without targeting PFK to the membrane after transfection of mutant CAV‐1. We propose that an interaction of CAV‐1 with PFK exists through interaction of the CSD with a binding motif in PFK. Support by NIH DK 60668.

Effects on kidney filtration rate by agmatine requires activation of ryanodine channels for nitric oxide generation

Agmatine, decarboxylated arginine, is produced in the kidney and can increase nephron and kidney filtration rate via renal vasodilatation and increases in plasma flow. This increase in filtration rate after agmatine is prevented by administration of nitric oxide synthase (NOS) inhibitors. In endothelial cells, agmatine-stimulated nitrite production is accompanied by induction of cytosolic calcium. NOS activity requires calcium for activation; however, the source of this calcium remains unknown. Ryanodine receptor (RyR) calcium-activated calcium release channels are present in the kidney cortex, and we evaluated if RyR contributes to the agmatine response. Agmatine microperfused into Bowman's space reversibly increases nephron filtration rate (SNGFR) by approximately 30%. cADP-ribose (cADPR) regulates RyR channel activity. Concurrent infusion of agmatine with the cADPR blocker 8-bromo-cADPR (2 microM) prevents the increase in filtration rate. Furthermore, direct activation of the RyR channel with ryanodine at agonist concentrations (5 microM) increases SNGFR, and, like agmatine, this increase is prevented by administration of N(G)-monomethyl-l-arginine, a nonselective NOS blocker. We demonstrate that agmatine does not elicit ADPR cyclase activity in vascular smooth muscle membranes and does not directly affect RyR calcium channel responses using sea urchin egg homogenates. These results imply interplay between endothelial cell cADPR/RyR/Ca(2+)/NO and the cADPR/RyR/Ca(2+) pathways in vascular smooth muscle cells in arterioles in the regulation of kidney filtration rate. In conclusion, we show that agmatine-induced effects require activation of cADPR and RyR calcium release channels for NO generation, vasodilation, and increased filtration rate.

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA 2 value of 9.17 ± 0.01 ( n = 5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 μM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [ 125I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The p Ki of MA-2029 was 8.58 ± 0.04 in the rabbit colon homogenate ( n = 4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 μg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 μg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.

Purinergic (P2) Receptor Control of Lower Genitourinary Tract Function and New Avenues for Drug Action: An Overview

Micturition, penile erection, contraction of prostatic smooth muscle, peristalsis of the male excurrent duct system and lumbosacral spinal cord neurotransmission all require adenosine 5'-triphosphate (ATP) activity and this likely involves purinergic (P2) receptors. P2 receptors are categorized as either ligand-gated ionotropic P2X or metabotropic G-protein-coupled P2Y subtypes. In the urinary bladder, purinergic receptor mechanisms are involved in both motor and sensory function. In the prostate, P2X1-receptors, which mediate contraction, are present in the fibromuscular stroma while G protein-coupled P2Y purinoceptors have a wide range of actions in prostate cancer. In the excretory ducts of the testis (ductus epididymidis, vas deferens and its associated seminal vesicles), heavy immunostaining for P2X1 and P2X2 subtypes is detected in the membranes of smooth muscle, suggesting their role in sperm transport and ejaculation. In the penis, intense P2X1 and weak P2X2 immunoreactivity are observed in smooth muscle of blood vessels and the corpus cavernosum, implying their participation in detumescence. Human corporal cavernosum stimulation induces relaxation of P2Y purinoceptors. Targeting of extracellular or intracellular P2X and/or P2Y receptor signaling pathways holds promise in affecting the lower genitourinary tract system. Our advancing knowledge about purine agonists and their pharmacologic benefits in erectile, ejaculatory, urinary bladder and prostatic hyperplasia may service clinical problems in the near future.