Mechanisms Of Smooth Muscle Contraction Research Articles

Death-associated protein kinase 3 regulates the myogenic reactivity of cerebral arteries.

What is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system. The vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

The Role of Tyrosine Kinase in Ca^(2+)-Independent Contraction of the Ropivacaine on Rat Aortic Smooth Muscle

The tyrosine kinase signaling pathway plays an important role in the mediation of Ca²⁺ independent mechanisms of smooth muscle contraction. Several components of this pathway, including protein kinase C (PKC), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and Rho-kinase are involved in Ca²⁺ independent mechanisms. Whether the tyrosine kinase pathway mediates vasoconstriction induced by the anesthetic ropivacaine remains unclear. The present study was designed to examine the role of tyrosine kinase in ropivacaine-induced, Ca²⁺-independent contraction of rat aortic smooth muscle. The effects of tyrosine kinase inhibitor on ropivacaine-induced contractile response were observed by isometric force measurement. The protein tyrosine phosphorylation, PKC, p44/42 MAPK, and membrane translocation of Rho-kinase were examined by Western blotting. Ropivacaine induced a concentration-dependent contractile response, and showed a number of effects on protein tyrosine phosphorylation. In this study, phosphorylation levels were shown to increase at lower concentrations of ropivacaine, but the levels decline at higher concentrations in rat aortic rings attenuated by the tyrosine kinase inhibitor genistein in a concentration-dependent fashion. Ropivacaine-induced phosphorylation of PKC and p44/42 MAPK and Rho-kinase membrane translocation were also significantly attenuated by genistein in similar decreasing manner as the PKC inhibitor bisindolylmaleimide I (Bis I) and the Rho-kinase inhibitor, Y27632, but to a lesser degree than that by the p44/42 MAPK inhibitor, PD 098059. Our results showed that the ropivacaine-induced, Ca²⁺ independent-mediated contraction of rat aortic smooth muscle is, in part, regulated by tyrosine kinase-catalyzed protein tyrosine phosphorylation.

A Review of Signal Transduction in Mechanisms of Smooth Muscle Contraction and Its Relevance for Specialized Physical Therapy

A Review of Signal Transduction in Mechanisms of Smooth Muscle Contraction and Its Relevance for Specialized Physical Therapy

Phosphorylation analysis of contractile regulatory proteins in smooth muscle by using phos-tag SDS electrophoresis

Activation of myosin by phosphorylation has been implicated in regulation of smooth muscle contractions. The ability to analyze myosin phosphorylation is crucial for better understanding of the regulatory mechanisms of smooth muscle contraction. In myosin phosphorylation analysis, it is important to quantify the ratio between phosphorylated and unphosphorylated myosin because those can have opposite effects on contractions. For this reason, urea/glycerol-PAGE and 2-D electrophoresis in combination with Coomassie blue staining or Western blotting are commonly used to separate and quantify myosin based on its phosphorylated state. However, those conventional techniques are not sensitive enough to analyze minute smooth muscles, such as terminal resistant arterioles, partly because of a poor separation in electrophoresis with a small sample. Recently we overcame this problem by introducing a phos-tag technique, and achieved picogram sensitivity. In this article, the sensitive phosphorylation quantification method and application of phos-tag electrophoresis to smooth muscle physiology research are reviewed.

Regulatory mechanism of smooth muscle contraction studied with gelsolin-treated strips of taenia caeci in guinea pig

The potential roles of the regulatory proteins actin, tropomyosin (Tm), and caldesmon (CaD), i.e., the components of the thin filament, in smooth muscle have been extensively studied in several types of smooth muscles. However, controversy remains on the putative physiological significance of these proteins. In this study, we intended to determine the functional roles of Tm and CaD in the regulation of smooth muscle contraction by using a reconstitution system of the thin filaments. At appropriate conditions, the thin (actin) filaments within skinned smooth muscle strips of taenia caeci in guinea pigs could be selectively removed by an actin-severing protein, gelsolin, without irreversible damage to the contractile apparatus, and then the thin filaments were reconstituted with purified components of thin filaments, i.e., actin, Tm, and CaD. We found that the structural remodeling of actin filaments or thin filaments was functionally linked to the Ca(2+)-induced force development and reduction in muscle cross-sectional area (CSA). That is, after the reconstitution of the gelsolin-treated skinned smooth muscle strips with pure actin, the Ca(2+)-dependent force development was partially restored, but the Ca(2+)-induced reduction in CSA occurred once. In contrast, the reconstitution with actin, followed by Tm and CaD, restored not only the force generation but also both its Ca(2+) sensitivity and the reversible Ca(2+)-dependent reduction in CSA. We confirmed that both removal of the thin filaments by gelsolin treatment and reconstitution of the actin (thin) filaments with Tm and CaD caused no significant changes in the level of myosin regulatory light chain phosphorylation. We thus conclude that Tm and CaD are necessary for the full regulation of smooth muscle contraction in addition to the other regulatory systems, including the myosin-linked one.

Disturbance of smooth muscle regulatory function by Eisenia foetida toxin lysenin: Insight into the mechanism of smooth muscle contraction

Lysenin, a toxin present in the coelomic fluid of the earthworm Eisenia foetida, is known to cause a long-lasting contraction of rat aorta smooth muscle strips. We addressed the mechanisms underlying its action on smooth muscle cells and present the first report demonstrating a completely new property of lysenin unrelated to its basic sphingomyelin-binding ability. Here we report lysenin enhancement effect on smooth muscle actomyosin ATPase activity and the ability of networking the actin filaments. The maximum enhancement of the ATPase activity of actomyosin at 120 mM KCl was observed at a molar ratio of lysenin to actin of about 1:10 5, while at 70 mM KCl at the ratio of about 1:10 6. The effect of lysenin became most pronounced only when both smooth muscle regulatory proteins, tropomyosin and caldesmon, were present. Co-sedimentation experiments indicated that lysenin did not displace neither tropomyosin nor caldesmon from the thin filament. Thus, the lysenin-dependent abolishment of the inhibitory effect of caldesmon on the ATPase activity was related rather to the modification of the filament structure. The ability of the toxin to exert its stimulatory effect at extremely low concentrations (as low as one molecule of lysenin per 10 6 actin molecules) may result from the long-range cooperative transitions in the entire thin filament with an involvement of smooth muscle tropomyosin, while the role of caldesmon may be limited exclusively to the inhibition of ATPase activity.

Sex hormones, Vascular Function and the Outcome of Hormone Replacement Therapy in Cardiovascular Disease

Cardiovascular disease is more common in men and post-menopausal women than premenopausal women, suggesting that female sex hormones have vascular benefits. Cytosolic/nuclear estrogen and progesterone receptors mediate genomic transcriptional effects that stimulate endothelial cell growth and inhibit smooth muscle proliferation. Sex hormone receptors on the plasma membrane trigger nongenomic stimulation of endothelium-dependent nitric oxide-cyclic (c)GMP, prostacyclin-cAMP and hyperpolarizing vascular relaxation pathways, as well as inhibition of [Ca2+](i), protein kinase C and Rho-kinase-dependent mechanisms of smooth muscle contraction. Despite the vasodilator effects of sex hormones, the Heart and Estrogen/progestin Replacement Study (HERS), HERS-II and Women's Health Initiative clinical trials have shown minimal benefits of hormone replacement therapy (HRT) in post-menopausal cardiovascular disease. The prospect of HRT relies on further mechanistic analysis of the vascular effects of natural sex hormones and phytoestrogens, and the identification of specific estrogen receptor modulators. Androgens have vascular effects, and modulators of the estrogen/testosterone ratio could provide better HRT combinations. The timing/duration and the type, dose and route of administration of HRT should be customized according to the subject's age and pre-existing cardiovascular condition, thereby enhancing the outcome of HRT in cardiovascular disease.

The vascular endothelin system in hypertension--recent patents and discoveries.

The discovery of endothelin two decades ago has now evolved into an intricate vascular endothelin (ET) system. Several ET isoforms, receptors, signaling pathways, agonists, antagonists, and clinical applications have been identified and documented in first-rate patents. The role of ET as one of the most potent endothelium-derived vasoconstricting factors is now complemented by a newly discovered role in vascular relaxation. ET synthesis is initiated by the transcription of ET genes in endothelial cells and the generation of the gene products preproET and big ET, which are further cleaved by specific ET converting enzymes into ET-1, -2, -3 and -4 isoforms. ET isoforms bind with different affinities to ET(A) and ET(B2) receptors in vascular smooth muscle, and stimulate [Ca(2+)](i), protein kinase C, mitogen-activated protein kinase and other signaling mechanisms of smooth muscle contraction, growth and proliferation. ET also binds to endothelial ET(B1) receptors, which mediate the release of vasodilator substances such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. Endothelial ET(B1) receptors may also function in ET re-uptake and clearance. Although the effects of ET on vascular function and growth are well-recognized, the role of ET and its receptors in the regulation of blood pressure and in the pathogenesis of hypertension is not clearly established. Salt-dependent hypertension in experimental animals and some forms of moderate to severe hypertension in human may show elevated levels of plasma or vascular ET; however, other forms of hypertension show normal ET levels. The currently available ET receptor antagonists reduce blood pressure in some forms of experimental hypertension. Careful examination of recent patents may identify more effective and specific modulators of the vascular ET system for clinical use in human hypertension.

Vascular Endothelin and Hypertension - From Receptors to Medicine

The last two decades have witnessed great advances in our understanding of the role of the vascular endothelin (ET) system in the control of blood pressure. The role of ET as one of the most potent endothelium-derived vasoconstrictors is now complemented with a newly discovered role in vascular relaxation. The transcription of the ET genes in endothelial cells leads to the formation of preproET and big ET, which are further metabolized by ET converting enzymes into ET-1, -2, -3 and -4 isoforms. ET isoforms bind with different affinities to ETA and ETB2 receptors in vascular smooth muscle, and stimulate [Ca2+]i and other signaling mechanisms of smooth muscle contraction and growth. ET also binds to endothelial ETB1 receptors, which are coupled to increase production of vasodilator substances such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor, and may also function in ET clearance. Despite the prominent effects of ET on vascular function and growth, the role of ET and its receptors in the regulation of blood pressure and in the pathogenesis of hypertension has not been clearly established. Some forms of moderate to severe hypertension in human and salt-dependent hypertension in experimental animals may show elevated levels of plasma or vascular ET; however, other forms of hypertension may show normal levels. Also, while ET antagonists could reverse some forms of experimental hypertension, their potential use in medicine needs to be carefully examined particularly with regard to their effectiveness and specificity.

Matrix metalloproteinase-specific inhibition of Ca 2+ entry mechanisms of vascular contraction

Abdominal aortic aneurysm (AAA) is a common disease with as yet unclear cause. Increased matrix metalloproteinase (MMP) levels in the plasma and aorta are a consistent finding in AAA. Although the role of MMPs in AAA has largely been attributed to degradation of the extracellular matrix proteins, the effects of MMPs on the mechanisms of aortic contraction are unclear. The purpose of this study was to test the hypothesis that MMPs promote aortic dilation by inhibiting the Ca2+ mobilization mechanisms of smooth muscle contraction. Isometric contraction and 45Ca2+ influx were measured in aortic strips isolated from male Sprague-Dawley rats treated or not treated with MMP-2 and MMP-9. In normal Krebs solution (2.5 mmol/L Ca2+ ) phenylephrine (10-5 mol/L) caused contraction of the aortic strips, which was significantly inhibited (P < .05) by MMP-2 (maximum, 48.9% +/- 5.0%) and to a greater extent by MMP-9 (maximum, 69.8% +/- 6.2%). The MMP-induced inhibition of phenylephrine contraction depended on concentration and time. The inhibitory effects of MMPs on phenylephrine contraction were reversible. In Ca2+ -free (2 mmol/L ethylene glycol bis[beta-aminoethyl ether]-N,N,N',N'-tetraacetic acid) Krebs solution phenylephrine caused a small contraction that was not inhibited by MMP-2 or MMP-9, which suggests that MMPs do not inhibit Ca2+ release from the intracellular stores. Membrane depolarization with 96 mmol/L of potassium chloride, which stimulates Ca2+ entry from the extracellular space, caused a time-dependent and reversible contraction, which was inhibited by MMP-2 and MMP-9. Histologic studies of MMP-treated tissues stained with hematoxylin-eosin or Verhoeff stain for elastin confirmed the absence of degradation of the extracellular matrix. MMP-2 and MMP-9 also caused significant inhibition of 45Ca2+ influx induced by phenylephrine and potassium chloride. These data suggest that MMP-2 and MMP-9 promote aortic dilation by inhibiting the Ca2+ entry mechanism of vascular smooth muscle contraction. Abdominal aortic aneurysm (AAA) is a slow and progressive disease. The late stages of AAA are characterized by degenerative changes in the extracellular matrix and smooth muscle components of the aortic wall. The present study describes novel inhibitory effects of matrix metalloproteinase (MMP) on the Ca2+ entry mechanisms of aortic smooth muscle contraction, even in the absence of extracellular matrix degradation. The MMP-induced inhibition of aortic contraction may further explain the role of increased MMP activity particularly during the early development of AAA. Chronic exposure to MMPs may lead to protracted inhibition of aortic contraction, progressive aortic dilation, and aneurysm formation. MMP-9 is a more potent inhibitor of aortic contraction than MMP-2, consistent with a more dominant role in AAA. Restoration and preservation of smooth muscle contractile function by specific inhibitors of MMPs may represent a new strategy in preventing the progression of small AAA.

Age-Related Reduction in Estrogen Receptor–Mediated Mechanisms of Vascular Relaxation in Female Spontaneously Hypertensive Rats

Hypertension increases with aging, and changes in vascular estrogen receptors (ERs) may play a role in age-related hypertension in women. We tested whether age-related increases in blood pressure in female spontaneously hypertensive rats (SHRs) are associated with reduction in amount and/or vascular relaxation effects of estrogen and ER. Arterial pressure and plasma estradiol were measured in adult (12 weeks) and aging (16 months) female SHRs, and thoracic aorta was isolated for measurement of active stress, 45Ca2+ influx, and ERs. Arterial pressure was greater and plasma estradiol was less in aging females than in adult females. In aorta of adult females, Western blots revealed alpha- and beta-ERs that were slightly reduced in aging rats. In endothelium-intact vascular strips, phenylephrine (Phe; 10(-5) mol/L) caused greater active stress in aging rats (9.3+/-0.2) than in adult rats (6.2+/-0.3x10(4) N/m2). 17beta-estradiol (E2) caused relaxation of Phe contraction and stimulation of vascular nitrite/nitrate production, which was reduced in aging rats. In endothelium-denuded strips, E2 still caused relaxation of Phe contraction, which was smaller in aging rats than adult rats. KCl (51 mmol/L), which stimulates Ca2+ influx, produced greater active stress in aging rats (9.1+/-0.3) than in adult rats (5.9+/-0.2x10(4) N/m2). E2 caused relaxation of KCl contraction and inhibition of Phe- and KCl-induced 45Ca2+ influx, which were reduced in aging rats. Thus, aging in female SHR is associated with reduction in ER-mediated NO production from endothelial cells and decrease in inhibitory effects of estrogen on Ca2+ entry mechanisms of smooth muscle contraction. The age-related decrease in ER-mediated vascular relaxation may explain the increased vascular contraction and arterial pressure associated with aging in females.

Second International Symposium “Smooth Muscle Physiology and Biophysics”

The 2nd International Symposium on Smooth Muscle Physiology and Biophysics is organized jointly by the Bogomolets Institute of Physiology of the National Academy of Sciences of Ukraine (Kyiv, Ukraine), St. George’s Hospital Medical School of London University (Great Britain), and Taras Shevchenko National Kyiv University with support from the National Academy of Sciences of Ukraine, All-Ukraine Public Organization “Association of Pharmacologists of Ukraine,” The Wellcome Trust (Great Britain), and The Physiological Society (Great Britain). It will be held at the Bogomolets Institute of Physiology on October 28-31, 2003. The 1st Kyiv symposium “Smooth Muscle Physiology” was carried out 30 years ago in the tense international atmosphere of the cold war. However, many leading scientists were able to come from abroad, and the meeting seemed to open a window for cooperation between scientists from the USSR and their western colleagues. Today, the situation is entirely different, as many international research projects are conducted at the Bogomolets Institute, while Ukrainian scientists are performing research at universities throughout the world. The symposium will include lectures presented by prominent scientists from many countries on a wide range of smooth muscle physiology, biophysics, and pharmacology topics, including ionic mechanisms of electrical excitation, molecular organization, and biophysical properties of ion channels, neural regulation of smooth muscle motility, receptor-gated ion channels, mechanisms of action of neurotransmitters, calcium signals and active ion transport, excitationcontraction coupling, and mechanics of smooth muscle contraction. Twenty-five invited talks and short communications will be presented in seven sessions, namely: “Molecular Components of Cationic Channels: TRP and SOC,” “Neuronal Functions and Neurotransmitters,” “Endothelium and Smooth Muscle Function,” “Spontaneous Activity in Smooth Muscles,” “Responses of Smooth Muscle to Neurotransmitters,” “Regulation of Smooth Muscle Contraction,” and “Emerging Roles of Non-Smooth Muscle Cells in Smooth Muscle Tissues.” Two poster sessions, which will include about 50 research communications, are also planned. The program of the symposium will, thus, reflect the diversity of problems, techniques, and achievements of modern smooth muscle physiology and biophysics. We are hoping that the meeting will take place in a friendly and stimulating atmosphere of productive scientific exchange, which will enable young researchers to discuss problems and questions of modern smooth muscle physiology and biophysics with distinguished experts in this field. The Organizing Committee is happy to announce that the present symposium will coincide with the 75th birthday of Professor Mykhailo F. Shuba, who is one of the founders of contemporary smooth muscle physiology. We congratulate him on this event and wish Prof. Shuba good health and successful continuation of his important research.

Testosterone and coronary vascular tone: implications in coronary artery disease.

The greater incidence of coronary artery disease in men compared to women has often suggested possible harmful effects of male sex steroids that could promote coronary atherogenesis and vasoconstriction. However, antiatherogenic and coronary vasodilator effects of testosterone have also been suggested. The interaction of testosterone (T) with its specific receptors may trigger not only long-term genomic effects, but also acute non-genomic vasodilator responses. Testosterone may activate the endothelium and stimulate the nitric oxide-cGMP and/or the hyperpolarization-mediated vascular relaxation pathway. T may also inhibit the signaling mechanisms of smooth muscle contraction such as [Ca2+]i and protein kinases. The T-induced stimulation of endothelium-dependent mechanisms of vascular relaxation and inhibition of the mechanisms of coronary smooth muscle contraction represent potential beneficial effects of T against coronary artery disease.

Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle.

The inhibition of myosin light chain phosphatase (MLCP) enhances smooth muscle contraction at a constant [Ca2+]. There are two components, myosin-binding subunit of MLCP (MBS) and CPI17, thought to be responsible for the inhibition of MLCP by external stimuli. The phosphorylation of MBS at Thr-641 and of CPI17 at Thr-38 inhibits the MLCP activity in vitro. Here we determined the changes in the phosphorylation of MBS and CPI17 after agonist stimulation in intact as well as permeabilized smooth muscle strips using phosphorylation-site-specific antibodies as probes. The CPI17 phosphorylation transiently increased after agonist stimulation in both alpha-toxin skinned and intact fibres. The time course of the increase in CPI17 phosphorylation after stimulation correlated with the increase in myosin regulatory light chain (MLC) phosphorylation. The increase in CPI17 phosphorylation was significantly diminished by Y27632, a Rho kinase inhibitor, and GF109203x, a protein kinase C inhibitor, suggesting that both the protein kinase C and Rho kinase pathways influence the change in CPI17 phosphorylation. On the other hand, a significant level of MBS phosphorylation at Thr-641, an inhibitory site, was observed in the resting state for both skinned and intact fibres and the agonist stimulation did not significantly alter the MBS phosphorylation level at Thr-641. While the removal of the agonist markedly decreased MLC phosphorylation and induced relaxation, the phosphorylation of MBS was unchanged, while CPI17 phosphorylation markedly diminished. These results strongly suggest that the phosphorylation of CPI17 plays a more significant role in the agonist-induced increase in myosin phosphorylation and contraction of smooth muscle than MBS phosphorylation in the Ca2+-independent activation mechanism of smooth muscle contraction.

Calcium-independent activation of the contractile apparatus in smooth muscle of mouse aorta by protein phosphatase inhibition.

Smooth muscle contraction is primarily regulated by reversible phosphorylation of the 20-kDa light chains of myosin (MLC(20)) involving Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK) and serine/threonine protein phosphatases (PP). The aim of this study was to investigate the effects of the protein phosphatases (PP) type 1 (PP1) and type 2A (PP2A) inhibitor cantharidin (Cant), its structural analogue endothall (ETA) and microcystin LR (MC) on force of contraction and MLC(20)-phosphorylation in arterial smooth muscle of mouse aorta. Cant increased force of contraction and MLC(20)-phosphorylation in intact arterial rings of mouse aorta in the presence of Ca(2+) whereas ETA and MC were ineffective under the same experimental conditions. In contrast, all compounds induced contraction and led to enhanced MLC(20)-phosphorylation in nominally Ca(2+)-free solution in fibers of mouse aorta permeabilised (skinned) with Triton X-100. In addition, Western blot analysis revealed that skinning of mouse aorta did not result in a loss of PP1 and PP2A compared to intact rings. Thus, both PP must be tightly bound to structural proteins, e.g. myosin. The findings indicate a Ca(2+)-independent mechanism of smooth muscle contraction involving inhibition of PP1- and/or PP2A-activities leading to enhanced force and MLC(20)-phosphorylation of arterial smooth muscle.

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure.

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.

Role of elastic fibers in cooling-induced relaxation

The objective of this work was to confirm the main role of elastic fibers in differing responses of certain vessels during cooling from 37 to 8 °C. Previous results have shown that the nature of the vessel (conduit vessel vs muscular vessel) determines the different behavior (dilatation vs contraction) of isolated vessel segments when temperature decreases from 37 to 8 °C. In this work, it has been demonstrated that vessels with a great amount of elastic fibers show a dilatation when cooling. On the other hand, muscular vessels with fewer elastic fibers, such as the renal artery, undergo a contraction. The output of calcium from intracellular stores causes contraction of the renal artery during cooling. In this vessel, vasodilatation occurs only when mechanisms of smooth muscle contraction are inactive, as is the case with vessels that have undergone a cold storage period of 48 h. The results presented in this work confirm that there are two main effects, which directly depend on the vessel origin. In conduit arteries, the decrease of temperature induces a vascular relaxation, dependent on the elastic component of the vessel wall. In muscular vessels, the predominant effect is cooling-induced contraction due to an increase of intracellular calcium. This cooling-induced contraction needs the vessel to be in optimal conditions with an active metabolism of the muscular cells. These results are a crucial issue in the sense of explaining several biomedical mechanisms where hypothermia is implicated. The type of vessel implicated in procedures, such as isolated organ perfusion, extracorporeal circulation, and bypass surgery, must be taken into account.

Enhanced [Ca 2+ ] I and Contractility of Renal Arterial Smooth Muscle in a Rat Model of Hypertension Produced by Reduction of Uterine Perfusion Pressure in Late Pregnancy

P147 Reduction of uterine perfusion pressure during late pregnancy has been suggested to trigger increases in renal vascular resistance and pregnancy-induced hypertension; however, the cellular mechanisms involved are unclear. We investigated whether reduction of uterine perfusion pressure in late pregnancy is associated with increased [Ca 2+ ] i and contractility of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of late pregnant Sprague-Dawley rats and a hypertensive pregnant rat model produced by chronic reduction in uterine perfusion pressure (RUPP). The cells were loaded with fura-2 and cell length and [Ca 2+ ] i were measured. In cells of pregnant rats incubated in Hank’s solution (1 mmol/L Ca 2+ ), resting [Ca 2+ ] i was 62±3 nmol/L and cell length was 69±3 μm. In RUPP rats, resting [Ca 2+ ] i (101±3 nmol/L) was significantly greater and cell length (53±2 μm) was shorter than pregnant rats. In pregnant rats, angiotensin II (AII, 10 -7 mol/L) caused transient increase in [Ca 2+ ] i to 412±25 nmol/L followed by a maintained increase to 158±15 nmol/L, and 23±2% cell contraction. In RUPP rats, the AII-induced [Ca 2+ ] i transient (434±22 nmol/L) was not significantly different from pregnant rats, but the maintained [Ca 2+ ] i was significantly elevated to 198±7 nmol/L and cell contraction was increased to 33±3%. In pregnant rats, the Ca 2+ channel agonist Bay K8644 (1 μmol/L) caused maintained increase in [Ca 2+ ] i to 292±12 nmol/L and 31% contraction that were significantly enhanced in RUPP rats. In Ca 2+ -free (2 mmol/L EGTA) Hank’s, AII- and caffeine (10 mmol/L)-induced [Ca 2+ ] i transient and cell contraction were not significantly different between pregnant and RUPP rats suggesting no difference in Ca 2+ release from intracellular stores. The enhanced basal, maintained AII- and Bay K8644-induced [Ca 2+ ] i and cell contractility in RUPP rats suggest enhanced Ca 2+ entry mechanisms of smooth muscle contraction in resistance renal arteries and may, in part, explain the increased renal vascular resistance associated with pregnancy-induced hypertension.

The Frequency Response of Smooth Muscle Stiffness during Ca 2+-Activated Contraction

To investigate the mechanism of smooth muscle contraction, the frequency response of the muscle stiffness of single β-escin permeabilized smooth muscle cells in the relaxed state was studied. Also, the response was continuously monitored for 3 min from the beginning of the exchange of relaxing solution to activating solution, and then at 5-min intervals for up to 20 min. The frequency response (30 Hz bandwidth, 0.33 Hz (or 0.2 Hz) resolution) was calculated from the Fourier-transformed force and length sampled during a 3-s (or 5-s) constant-amplitude length perturbation of increasing-frequency (1–32 Hz) sine waves. In the relaxed state, a large negative phase angle was observed, which suggests the existence of attached energy generating cross-bridges. As the activation progressed, the muscle stiffness and phase angle steadily increased; these increases gradually extended to higher frequencies, and reached a steady state by 100 s after activation or ∼40 s after stiffness began to increase. The results suggest that a fixed distribution of cross-bridge states was reached after 40 s of Ca 2+ activation and the cross-bridge cycling rate did not change during the period of force maintenance.

Tyrosine kinase inhibitors suppress prostaglandin F 2α-induced phosphoinositide hydrolysis, Ca 2+ elevation and contraction in iris sphincter smooth muscle

We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F 2α- and carbachol-induced inositol-1,4,5-trisphosphate (IP 3) production, [Ca 2+] i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F 2α and carbachol induced contraction in a concentration-dependent manner with EC 50 values of 0.92×10 −9 and 1.75×10 −8 M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F 2α, but not those evoked by carbachol, on IP 3 accumulation, [Ca 2+] i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F 2α analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 μM) markedly reduced (by 67%) prostaglandin F 2α-stimulated increase in [Ca 2+] i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC 50 of 82 μM and increased IP 3 generation in a concentration-dependent manner with an EC 50 of 90 μM. The effects of vanadate were abolished by genistein (10 μM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F 2α- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca 2+] i evoked by prostaglandin F 2α. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F 2α receptor activation and increases in intracellular Ca 2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.