Strips Of Carotid Arteries Research Articles

Immunoneutralization of human angiotensin-(1-12) with a monoclonal antibody in a humanized model of hypertension

We engineered a monoclonal antibody (mAb) against the human C-terminus of angiotensin-(1–12) [h-Ang-(1–12)] and performed a biochemical characterization in concert with direct in vivo and ex vivo (carotid artery strips) assessments of h-Ang-(1–12) vasoconstrictor activity in 78 (36 females) transgenic rats expressing the human angiotensinogen gene [TGR(hAGT)L1623] and 26 (10 female) Sprague Dawley (SD) controls. The mAb shows high specificity in neutralizing angiotensin II formation from h-Ang-(1–12) and did not cross-react with human and rat angiotensins. Changes in arterial pressure and heart rate in Inactin® hydrate anesthetized rats were measured before and after h-Ang-(1–12) injections [dose range: 75–300 pmol/kg i.v.] prior to and 30–60 minutes after administration of the h-Ang-(1–12) mAb. Neutralization of circulating Ang-(1–12) inhibited the pressor action of h-Ang-(1–12), prevented Ang-(1–12) constrictor responses in carotid artery rings in both SD and TGR(hAGT)L1623 rats, and caused a fall in the arterial pressure of male and female transgenic rats. The Ang-(1–12) mAb did not affect the response of comparable dose-related pressor responses to Ang II, pre-immune IgG, or the rat sequence of Ang-(1–12). This h-Ang-(1–12) mAb can effectively suppress the pressor actions of the substrate in the circulation of hypertensive rats or in carotid artery strips from both SD and transgenic rats. The demonstration that this Ang-(1–12) mAb by itself, induced a fall in arterial pressure in transgenic hypertensive rats supports further exploring the potential abilities of Ang-(1–12) mAb in the treatment of hypertension.

An investigation into the role of different constituents in damage accumulation in arterial tissue and constitutive model development

Carotid atherosclerotic plaque rupture is one of the leading causes of stroke. Treatments for atherosclerosis can induce tissue damage during the deployment of an intravascular device or through external tissue clamping during surgery. In this paper, a constituent specific study was performed to investigate the role of the ground matrix and collagen fibres of arterial tissue in response to supra-physiological loads. Cyclic mechanical tests were conducted on intact and collagenase-digested strips of porcine common carotid arteries. Using these tests, four passive damage-relevant phenomena were studied, namely (i) Mullins effect, (ii) hysteresis, (iii) permanent set and (iv) matrix failure and fibre rupture. A constitutive model was also developed to capture all of these damage-relevant phenomena using a continuum damage mechanics approach. The implemented constitutive model was fit to experimental results for both intact and digested samples. The results of this work demonstrate the important role of the ground matrix in the permanent deformation of the arterial tissue under high loads. Supra-physiological load-induced tissue damage may play a key role in vascular remodelling in arteries with atherosclerosis or following interventional procedures.

Sustained Contraction in Vascular Smooth Muscle by Activation of L-type Ca2+ Channels Does Not Involve Ca2+ Sensitization or Caldesmon.

Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca2+ channels (LTCC). This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544.

Endothelium-Dependent Vasorelaxation Effects of Rubus Coreanus extract on Rabbit Carotid Artery

This study was investigated to evaluate the vasorelaxant effect of Rubus coreanus(RC) extract on contracted rabbit carotid artery and its mechanism. To study the effect of RC extract on contracted rabbit carotid arterial strips, arterial strips with intact or damaged endothelium were used for experiment using organ bath. The pre-contracted arterial strips with norepinephrine(NE) or potassium chloride(KCl) was treated with various concentrations of an extract of RC(0.01, 0.03, 0.1, 0.3 and 1.0 ㎎/㎖). To determine the mechanisms of RC-induced vasorelaxant, RC extract was infused into contracted arterial rings which had been pretreated by indomethacin(IM), tetraethylammonium chloride(TEA), Nω-nitro-L-arginine (L-NNA), methylene blue(MB). And calcium chloride(Ca) 1 mM was infused into precontracted arterial ring induced by NE or KCl after treatment of RC extract in Ca<SUP>2+</SUP>-free krebs solution. Cytotoxic activity of RC extract on human umbilical vein endothelial cell(HUVEC) was measured by MTT assay, and nitric oxide(NO) prodution was measured by Griess reagent. RC extract revealed significant relaxation on NE-induced arterial contraction, but didn"t relax on KCl-induced arterial contraction. RC extract also had an effective relaxation to the intact endothelium arterial ring, but not the damaged endothelium arterial ring. Treatment of IM, TEA, L-NNA, MB reduced the relaxation of RC extract. Pretreatment of RC extract inhibited the contraction by influx of extracellular Ca<SUP>2+</SUP> in contracted arterial ring induced by NE, but it didn"t work the contraction by influx of extracellular Ca<SUP>2+</SUP> in contracted arterial ring induced by KCl in Ca<SUP>2+</SUP>-free krebs solution. RC extract increased nitric oxide production on HUVEC. This study indicated that the relaxation effect of RC extract on contracted rabbit carotid artery is related with NO-cGMP pathway, EDHF, prostacyclin.

Effects of reserpine and hydrallazine on isolated strips of carotid arteries.

Abstract Spirally cut sections of horse carotid artery have been found suitable for the qualitative testing of vasoconstrictors and their antagonists. With this preparation reserpine and hydrallazine non-specifically antagonise the contractions induced by a number of stimulant drugs, including adrenaline, noradrenaline, histamine, 5-hydroxytryptamine, acetylcholine, barium chloride and potassium chloride. Measurements of “self protection” by means of Furchgott's method but using strips of horse carotid arteries have shown that hydrallazine may possess an affinity for adrenergic receptors.

Antagonism to the actions of hydrallazine, reserpine, potassium cyanide, sodium azide and anoxia on arterial smooth muscle.

Abstract Many intermediates of carbohydrate, fat and protein metabolism give protection against the hydrallazine depression of acetylcholine, (-)-adrenaline, (-)-noradrenaline, histamine and 5-hydroxytryptamine-induced contractions of spirally cut strips of horse carotid arteries. Strips made anoxic behave in a manner similar to hydrallazine-treated strips. Inhibition of drug-induced contractions by potassium cyanide, iodoacetate and azide was of a different character from that caused by hydrallazine and anoxia. Few intermediates gave significant protection. Hydrallazine probably exerts its effect by a non-specific depression of metabolism rather than upon specific receptors. Reserpine depression of drug-induced contractions in artery strips was so persistent that experiments using intermediary metabolites could not be made.

THE RESPONSE TO TRANSMURAL STIMULATION OF ISOLATED ARTERIAL STRIPS AND ITS MODIFICATION BY DRUGS.

Abstract Transmural electrical stimulation of isolated aortic strips of rabbits, induced contractions of the muscle which were blocked by concentrations of cocaine and lignocaine which augmented the response to noradrenaline. The adrenergic neurone blocking agents bretylium, guanethidine and bethanidine also blocked selectively the response to transmural stimulation. With bretylium and guanethidine the responses to noradrenaline were augmented. α-Receptor blockade with piperoxan, phentolamine, phenoxybenzamine and dihydrogenated ergot alkaloids also blocked the responses to transmural stimulation. Cocaine in low concentration and dexamphetamine augmented the responses to electrical stimulation. They also delayed onset of block with adrenergic neurone blocking drugs and reversed this when it was already established. Hexamethonium and pentolinium, in concentrations which in other tissues are known to cause complete blockade of ganglionic transmission. had no effect on electrical stimulation. It is concluded that the responses to electrical stimulation are the result of excitation of post-ganglionic adrenergic axons in the walls of the arterial preparation. Strips of common carotid, superior mesenteric, renal, pulmonary and common iliac arteries of the rabbit gave responses to electrical stimulation similar to those of the aorta.

Influence of hyperthermia on carotid blood flow using 99mTc-HMPAO

Hyperthermia can be the result of many causes such as environmental conditions, brain tumors and infectious diseases. Since hyperthermia is very common, its role in causing stroke through a decrease in cerebral blood flow needed further emphasis. The aim of this study was to record cerebral blood flow in vitro by using isolated rabbit carotid artery strips and in-vivo using radioactive isotope scanning during temperature elevation. The recording of isometric tension in rabbit carotid artery strips in organ baths, and the scintigraphic cerebral imaging of technetium-99m-hexamethyl-propyleneamineoxime (99mTc-HMPAO) using Gamma camera, were acquired at control and higher body temperature by 4 degrees C. Blood pressure was measured through femoral artery and cerebral blood flow was measured through carotid artery. Elevating temperature by 4 degrees C induced reproducible contraction. During hyperthermia, the carotid artery contraction leads to a decrease in cerebral blood flow although the blood pressure did not decrease. The uptake of 99mTc-HMPAO in the brain was significantly reduced. This decrease in cerebral perfusion is regionally dependent, which is more in the frontal area, the cerebral hemispheres than the cerebellum. The decrease was 36+/-3, 37+/-2, 22+/-2%, respectively. Hyperthermia causes carotid artery contraction leading to decrease in cerebral blood flow, which was confirmed by 99mTc-HMPAO images. The decrease is regionally dependent. Since the blood pressure did not decrease by heating, the reduction in cerebral perfusion is mainly due to carotid contraction. The applied neck cooling may be considered as a promising therapeutic strategy for the hyperthermic patient to avoid brain damage. This can be achieved by external application of an ice-water-perfused neck collar.

Synergistic effects of ethanol and hyperthermia on carotid artery vasoconstriction

Heatstroke is a serious condition and clinical studies indicate that vascular stroke increases with excessive consumption of alcohol (ethanol). It was our objective to test the influence of ethanol on cerebral perfusion at normal and higher temperatures. Recording of isometric tension in rabbit carotid artery strips in organ baths with different concentrations of ethanol at 37 degrees C and during hyperthermia (39-43 degrees C) and scintigraphic cerebral imaging of a radioactive isotope in the control situation and during hyperthermia. Stepwise heating induced reproducible reversible graded contraction, proportional to temperature. At high concentrations (toxic levels), ethanol induced an increase in tension and heating potentiated these responses. Extracellular Mg(2+) potentiated both heat-induced contraction and ethanol-induced contraction while extracellular Ca(2+) had no effect on these responses. During hyperthermia and ethanol scintigraphic isotope uptake was reduced in cortical and cerebellar regions. Carotid artery vasomotor tone is temperature dependent and heating induces vasoconstriction. Alcohol (ethanol) at 37 degrees C elicited carotid artery contraction at high concentrations (toxic levels) but at any concentration during elevated temperature (39-43 degrees C). Ethanol potentiated the effect of hyperthermia-induced vasoconstriction and reduced cerebral perfusion as shown by radionuclide imaging. The synergistic effect of ethanol and hyperthermia may induce heat stroke and brain damage.

SiRNA knock down of casein kinase 2 increases force and cross-bridge cycling rates in vascular smooth muscle

Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction, suggesting that other regulatory mechanisms exist. One potentially important technique to study alternative sites of contractile regulation is the use of small interfering RNA (siRNA). The goal of this study was to determine whether siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein affects contractile regulation. To achieve this goal, we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a approximately 60% knockdown after 4 days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in cross-bridge cycling rates but not by a change in MLC phosphorylation levels. alpha-Toxin-permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium compared with control tissues. Our results demonstrate that siRNA is a viable technique with which to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and cross-bridge cycling by a MLC phosphorylation-independent pathway.

Hyperthermia-Induced Vasoconstriction of the Carotid Artery and the Role of Potassium Channels

Clinical experience and experimental studies have shown that hyperthermia can cause cerebral ischaemia and brain damage. By in vitro experiments with heating, we previously were able to induce carotid artery constriction. The objective of the present study was to clarify the mechanism of this thermal response. Isometric tension was recorded in rabbit carotid artery specimens using organ baths during stepwise temperature elevation. The heating responses were investigated at basal tone, in precontracted vessels, after blocking of adrenergic responses and administration of potassium (K)-channel activators and inhibitors. Stepwise heating of carotid artery strips from 37 degrees C to 47 degrees C induced reproducible graded contraction. The hyperthermic responses were not due to adrenergic stimulation, which were reduced and resistant to neurogenic blockade by tetrodotoxin. Heating-induced contractions were potentiated by the K-channel inhibitors tetraethylammonium, BaCl2, charybdotoxin, and the Na+/K+ ATPase inhibitor ouabain. Levcromakalim (BRL), a K+-channel activator, reduced heating induced contractions. Heating of carotid artery preparations induced reversible graded vasoconstriction proportional to temperature. The heating-induced contractions were not mediated by an adrenergenic process, but rather were due to inhibition of K+ channels, which increases Ca2+ entry. In vivo, this reaction may lead to a disturbance of autoregulation of cerebral blood flow and ischemia with brain damage.

Hyperthermia-induced vasoconstriction of the carotid artery, a possible causative factor of heatstroke.

Clinical and experimental studies indicate that hyperthermia can cause heatstroke with cerebral ischemia and brain damage. However, no study has examined the direct effects of heating carotid artery smooth muscle and tested the hypothesis that hyperthermia induces arterial vasoconstriction and, thereby, decreases cerebral blood flow. We recorded isometric tension of rabbit carotid artery strips in organ baths during stepwise temperature elevation. The heating responses were tested at basal tone, in norepinephrine- and KCl-precontracted vessels, and after electrical field stimulation. Stepwise heating from 37 degrees C to 47 degrees C induced reproducible graded contraction proportional to temperature. The responses could be elicited at basal tone and in precontracted vessels. Heating decreased the contractile responses to norepinephrine and electrical field stimulation but increased contraction to KCl. These responses were not eliminated by pretreatment with the neuronal blocker tetrodotoxin. Our results demonstrate that heating carotid artery preparations above 37 degrees C (normothermia) induced a reversible graded vasoconstriction proportional to temperature. In vivo this reaction may lead to a decrease in cerebral blood flow and cerebral ischemia with brain damage as in heatstroke. The heating-induced contraction is not mediated by a neurogenic process but is due to altered transcellular Ca2+ transport. Cooling, in particular of the neck area, therefore, should be used in the treatment of heatstroke.

Optimal dye concentration and irradiance for laser-assisted vascular anastomosis.

This investigation was done in order to find optimal indocyanine green (ICG) concentration and energy irradiance in laser vascular welding. Many studies have shown that laser tissue welding with albumin solder/ICG may be an effective technique in surgical reconstruction. However, there are few reports regarding optimal laser settings and concentrations of ICG within the albumin solder in laser-assisted vascular anastomosis. Porcine carotid artery strips (n = 120) were welded in end-to-end by diode laser with 50% albumin solder of 0.01, 0.1, and 1.0 mM ICG at irradiance of 27.7, 56.7, and 76.9 W/cm(2), respectively. Temperature was measured by inserting thermocouples outside and inside the vessel. Tensile strength and histology were studied. Temperature and strength of the anastomosis significantly decreased (all p < 0.05) with increasing ICG concentration at 56.7 W/cm(2). Histological study showed minimal thermal injury limited to adventitia and no appreciable difference between all groups. ICG concentration within solder is the most important factor affecting both vascular temperature and tensile strength. The optimal balance between strength and minimal thermal injury may be achieved primarily at 56.7 W/cm(2) and 0.01 mM ICG.

청상사화탕(淸上瀉火湯)이 토끼의 수축혈관에 미치는 영향

This study was undertaken to define the effect of ChungSangSaWhaTang on the norepinephrine-induced arterial contraction and the mechanism of ChungSangSaWhaTang-induced relaxation. In order to investigate the effect of ChungSangSaWhaTang on contracted rabbit carotid arterial strips, transverse strips with intact or damaged endothelium were used for the experiment using organ bath. To analyze the mechanism of ChungSangSaWhaTang-induced relaxation, ChungSangSaWhaTang extract infused into contracted arterial strips induced by norepinephrine after treatment of indomethacin, -nitro-L-arginine, or tetraethylammonium chloride. ChungSangSaWhaTang relax arterial strip with endothelium contracted by norepinephrine, but in the strips without endothelium, ChungSangSaWhaTang-induced relaxation was significantly inhibited. The endothelium-dependent relaxation induced by ChungSangSaWhaTang was decreased by the pretreatment of -nitro-L-arginine, but it was not observed in the strips pretreated with indomethacin or tetraethylammonium chloride. We suggest that ChungSangSaWhaTang may inhibit agonist-induced contraction through the formation of nitric oxide in the vascular endothelial cells.

Lactate depresses sarcolemmal permeability of Ca 2+ in intact arterial smooth muscle

Elevation of ambient lactate concentration has been shown to alter contractile reactivity of vascular smooth muscle. We tested the hypothesis that lactate affects the disposition of intracellular free Ca 2+. Porcine carotid artery strips were incubated in normal medium and in medium containing 10 mM sodium lactate or 10 mM sodium pyruvate. The rate and magnitude of contraction in response to K +-depolarization was depressed in lactate when compared to control. This was associated with a decrease in the onset and magnitude of the normal increase in free [Ca 2+] i, as reflected by fluorescence of fura-2. Pyruvate had no effect on these variables. Depression in [Ca 2+] i could not be attributed to a selective effect of lactate on pH i, membrane potential, or to enhanced superoxide production. Deletion of Ca 2+ from the incubation medium negated depression of contractile responsiveness produced by lactate when compared to control. Lactate had no effect on contraction induced by 100 μM norepinephrine, which releases intracellular stored Ca 2+. Thus, lactate inhibits arterial smooth muscle contraction by inhibiting influx of Ca 2+ across the sarcolemma.

The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin

Background. The heat shock-related protein (HSP) 20 is associated with actin and modulates smooth-muscle relaxation. We hypothesized that HSP20 mediates vasorelaxation via dynamic interactions with cytoskeletal proteins, such as actin, or actin binding proteins, such as α-actinin. Methods. Physiological responses of strips of bovine carotid artery were analyzed with a muscle bath. In other experiments, the arteries were homogenized, and imunoprecipitations were performed. Immunohistochemistry with anti-HSP20 and anti-actinin antibodies was used to determine co-localization of the two proteins. Results. Bovine carotid arteries contracted in response to serotonin and rapidly relaxed in response to forskolin. HSP20 co-immunoprecipitated with both actin and α-actinin, but not with HSP27 or paxillin. Immunostaining with HSP20 and α-actinin antibodies demonstrated that HSP20 and α-actinin co-localized. The amount of HSP20 that immunoprecipitated with α -actinin was markedly diminished in muscles that were treated with the vasorelaxant forskolin. Conclusions. HSP20 is associated with both actin and α-actinin. Activation of cyclic nucleotide-dependent signaling pathways leads to increases in the phosphorylation of HSP20 and a decrease in the association of HSP20 with α-actinin. These data suggest that phosphorylation of HSP20 may lead to relaxation of vascular smooth muscles through a dynamic association with cytoskeletal elements.

Effects of papaya seed extract and benzyl isothiocyanate on vascular contraction

To investigate their potentially toxic effects on mammalian vascular smooth muscle, pentane extracts of papaya seeds and the chief active ingredient in the extracts, benzyl isothiocyanate (BITC), were tested for their effects on the contraction of strips of dog carotid artery. BITC and the papaya seed extract caused relaxation when added to tissue strips that had been pre-contracted with phenylephrine (PE). Incubation of the tissue with papaya seed extract or BITC caused inhibition of contraction when the strips were subsequently contracted with KCl or PE. This relaxation and inhibition of contraction did not appear to be endothelium-dependent, as endothelium-denuded rings showed the same degree of relaxation or inhibition of contraction in response to the preparations/drugs as those with the endothelium intact. The effects of both BITC and the extract were irreversible, i.e., the tissue did not recover to normal contractile ability after extensive washing. Exposure of the tissue to the papaya seed extract caused slower relaxation of the tissue, compared to controls, both after contraction with PE and subsequent addition of carbachol (CCh), and after contraction with KCl and then washing. Calcium imaging studies using cultured endothelial cells showed strong influxes of Ca 2+ into the cells in response to addition of the papaya seed extract. We conclude that these extracts, when present in high concentration, are cytotoxic by increasing the membrane permeability to Ca 2+, and that the vascular effects of papaya seed extracts are consistent with the notion that BITC is the chief bio-active ingredient.

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats.

Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.

Inhibition of ERK attenuates force development by lowering myosin light chain phosphorylation.

Phosphorylation of the actin-associated protein caldesmon (CaD) by extracellular signal-regulated kinases (ERK1/2) is purported to participate in force maintenance by vascular smooth muscle. We examined the interrelationship among ERK1/2 activity, phosphorylation of the high molecular weight isoform of CaD (h-CaD) and the 20-kDa myosin light chain (LC(20)), and isometric force in strips of porcine carotid artery stimulated with endothelin-1 (ET-1; 50 nM). After an initial delay, ERK1/2 activity increased in parallel with ET-1-mediated force; h-CaD phosphorylation increased modestly. 2-(2'-Amino-3'-methoxyphenyl)-ox-anaphthalen-4-one (PD-098059; 50 microM), an ERK1/2 kinase inhibitor, significantly reduced basal ERK1/2 activity within 1 h, but only partially attenuated h-CaD phosphorylation at 3 h. The mechanisms underlying the temporal dissociation of ERK1/2 activity from h-CaD phosphorylation are unknown, but include the possibility that a kinase other than ERK1/2 phosphorylates h-CaD or, more likely, that phosphate turnover in h-CaD is very slow. PD-098059 partially inhibited the development of ET-1-stimulated force only in Ca(2+)-replete physiological saline solution, primarily by reducing LC(20) phosphorylation, yet had no effect on myosin light chain kinase in vitro. These inhibitory effects were most evident during the early phase of force production. The inhibitory effect of PD-098059 on force could not be correlated with a corresponding effect on ERK1/2-mediated h-CaD phosphorylation because force in arterial strips stimulated with ET-1 in the absence or presence of PD-098059 tended to approximate each other over time despite significant differences in the level of h-CaD phosphorylation. Force and LC(20) phosphorylation in response to KCl depolarization were unaffected by PD-098059. These results show that ERK1/2 may regulate force in arterial smooth muscle, but suggest that the mechanism for this effect is by inhibiting LC(20) phosphorylation.

Effects of flufenamic acid on smooth muscle of the carotid artery isolated from spontaneously hypertensive rats.

Endothelium-removed carotid artery strips from stroke-prone spontaneously hypertensive rats spontaneously developed a tonic myogenic contraction. Flufenamic acid reduced the resting tone observed during superfusion with Tyrode's solution, in a concentration-dependent manner. Flufenamic acid also inhibited contractions produced by high-K solutions in a concentration-dependent manner. The resting membrane potential of smooth muscle cells in the artery was around -32 mV, with occasional oscillatory potentials. Flufenamic acid hyperpolarized the membrane in a concentration-dependent manner. The voltage-dependent outward currents recorded in isolated cells with micropipettes filled with high-K+ solution (holding potential, -60 mV) were enhanced by flufenamic acid and inhibited by tetraethylammonium. When the recording micropipette was filled with high Cs to inhibit the K+-current, depolarizing step pulses evoked nifedipine-sensitive inward currents. Flufenamic acid inhibited the inward currents. These results indicate that flufenamic acid inhibits the spontaneous active tone of the carotid artery by inhibiting L-type Ca2+-channels and possibly by membrane hyperpolarization through activation of the voltage-dependent K+-channels.