Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac,NSC23766 and EHT1864

Background and Purpose

Medical therapy of lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) targets smooth muscle contraction in the prostate, or prostate growth. However, current therapeutic options are insufficient. Here, we investigated the role of Rac in the control of smooth muscle tone in human prostates and growth of prostate stromal cells.

Experimental Approach

Experiments were performed using human prostate tissues from radical prostatectomy and cultured stromal cells (WPMY-1). Expression of Rac was examined by Western blot and fluorescence staining. Effects of Rac inhibitors (NSC23766 and EHT1864) on contractility were assessed in the organ bath. The effects of Rac inhibitors were assessed by pull-down, cytotoxicity using a cell counting kit, cytoskeletal organization by phalloidin staining and cell growth using an 5-ethynyl-2′-deoxyuridine assay.

Key Results

Expression of Rac1–3 was observed in prostate samples from each patient. Immunoreactivity for Rac1–3 was observed in the stroma, where it colocalized with the smooth muscle marker, calponin. NSC23766 and EHT1864 significantly reduced contractions of prostate strips induced by noradrenaline, phenylephrine or electrical field stimulation. NSC23766 and EHT1864 inhibited Rac activity in WPMY-1 cells. Survival of WPMY-1 cells ranged between 64 and 81% after incubation with NSC23766 (50 or 100 μM) or EHT1864 (25 μM) for 24 h. NSC23766 and EHT1864 induced cytoskeletal disorganization in WPMY-1 cells. Both inhibitors impaired the growth of WPMY-1 cells.

Conclusions and Implications

Rac may be a link connecting the control of prostate smooth muscle tone with proliferation of smooth muscle cells. Improvements in LUTS suggestive of BPH by Rac inhibitors appears possible.     

ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human periodontal ligament fibroblasts via Rac1/Cdc42

Rho GTPase-activating protein (Rho-GAP) and Rho GDP dissociation inhibitor (Rho- GDI) are two main negative regulators of Rho GTPase. Our previous work has found that Rho-GDI and Rho GTPase are involved in the response of human periodontal ligament (PDL) cells to mechanical stress. However, whether Rho-GAP also has a role in this process remains unknown. Here, we attempted to find the Rho-GAP gene that may be involved in pathological stretch-induced apoptosis of PDL cells. Human PDL fibroblasts were exposed to 20% cyclic strain for 6 hours or 24 hours, after which the expression levels of ARHGAP10, ARHGAP17, ARHGAP21, ARHGAP24 and ARHGAP28 were determined. Results showed that ARHGAP17 expression decreased the most obviously after treatment of stretch. In addition, ARHGAP17 overexpression abolished 20% cyclic strain-induced apoptosis. Therefore, ARHGAP17 has an important role in pathological stretch-induced apoptosis of human PDL fibroblasts. Moreover, we found that ARHGAP17 overexpression also alleviated cyclic strain-induced activation of Rac1/Cdc42, a major downstream target of ARHGAP17. Furthermore, two Rac1 inhibitors, NSC23766 and EHT 1864, both attenuated ARHGAP17 knockdown-mediated apoptosis in human PDL fibroblasts. Collectively, our data demonstrate that ARHGAP17 inhibits pathological cyclic strain-induced apoptosis in human PDL fibroblasts through inactivating Rac1/Cdc42. This study highlights the importance of Rho signalling in the response of human PDL fibroblasts to mechanical stress.     

Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility

Objectives Lipopolysaccharide (LPS) has been shown to alter intestinal contractility. Toll‐like receptor 4 (TLR4), K⁺ channels and mitogen‐activated protein kinases (MAPKs) have been proposed to be involved in the mechanism of action of LPS. The aim of this study was to determine the role of TLR4, K⁺ channels and MAPKs (p38, JNK and MEK1/2) in the local effect of LPS on the acetylcholine (ACh)‐induced contractions in rabbit small intestine in vitro. Methods Segments of rabbit duodenum were suspended in the direction of longitudinal or circular smooth muscle fibres in a thermostatically controlled organ bath. Key findings LPS (0.3 µg/ml) reduced the contractions induced by ACh (100 µm ) in the longitudinal and circular smooth muscle of the duodenum after 90 min of incubation. Polymyxin (TLR4 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor) and U0126 (MEK1/2 inhibitor) antagonized the effects of the LPS on ACh‐induced contractions in duodenal smooth muscle. Incubation with the blockers of K⁺ channels, TEA, apamin, charybdotoxin, iberiotoxin, glibenclamide or quinine, did not reverse the effect of LPS on ACh‐induced contractions. Conclusions These results suggest that the effect of LPS on ACh‐induced contractions in the rabbit duodenum might be mediated by TLR4 and p38, JNK1/2 and MEK1/2 MAPKs.     

The Influences of G Proteins, Ca2+, and K+ Channels on Electrical Field Stimulation in Cat Esophageal Smooth Muscle

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin (1 µM) and atropine (1 µM). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of ''on'' and ''off'' contraction and the effects of G-proteins, phospholipase, and K⁺ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a G_i inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, G_s inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a K⁺ channel opener) decreased these contractions, and tetraethylammonium (TEA, K⁺_Ca channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type Ca²⁺ channel may be activated by G-protein α subunits. Furthermore, K⁺_Ca-channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of Ca²⁺ channel and to investigate the effects of other K⁺ channels on EFS-induced on and off contractions.     

Relaxing Responses to Hydrogen Peroxide and Nitric Oxide in Human Pericardial Resistance Arteries Stimulated with Endothelin‐1

In human pericardial resistance arteries, effects of the endothelium‐dependent vasodilator bradykinin are mediated by NO during contraction induced by K⁺ or the TxA₂ analogue U46619 and by H₂O₂ during contraction by endothelin‐1 (ET‐1), respectively. We tested the hypotheses that ET‐1 reduces relaxing effects of NO and increases those of H₂O₂ in resistance artery smooth muscle of patients with cardiovascular disease. Arterial segments, dissected from the parietal pericardium of 39 cardiothoracic surgery patients, were studied by myography during amplitude‐matched contractions induced by K⁺, the TXA₂ analogue U46619 or ET‐1. Effects of the NO donor Na‐nitroprusside (SNP) and of exogenous H₂O₂ were recorded in the absence and presence of inhibitors of cyclooxygenases, NO synthases and small and intermediate conductance calcium‐activated K⁺ channels. During contractions induced by either of the three stimuli, the potency of SNP did not differ and was not modified by the inhibitors. In vessels contracted with ET‐1, the potency of H₂O₂ was on average and in terms of interindividual variability considerably larger than in K⁺‐contracted vessels. Both differences were not statistically significant in the presence of inhibitors of mechanisms of endothelium‐dependent vasodilatation. In resistance arteries from patients with cardiovascular disease, ET‐1 does not selectively modify smooth muscle relaxing responses to NO or H₂O₂. Furthermore, the candidate endothelium‐derived relaxing factor H₂O₂ also acts as an endothelium‐dependent vasodilator.     

Xanthine‐analog,KMUP‐2, enhances cyclic GMP and K+ channel activities in rabbit aorta and corpus cavernosum with associated penile erection

The pharmacological properties of KMUP‐2 were examined in isolated rabbit aorta and corpus cavernosum smooth muscle (CCSM). KMUP‐2 caused relaxations that were attenuated by removed endothelium, high K⁺, and pretreatment with the soluble guanylate cyclase (sGC) inhibitors methylene blue (10 μM) and ODQ (1 μM), a NOS inhibitor, L‐NAME (100 μM), a K⁺ channel blocker TEA (10 mM), a K_ATP channel blocker glibenclamide (1 μM), a voltage‐dependent K⁺ channel blocker 4‐AP (100 μM), and the Ca²⁺‐dependent K⁺ channel blockers apamin (1 μM) and charybdotoxin (ChTX, 0.1 μM). The relaxant responses of KMUP‐2 (0.01, 0.05, 0.1 μM) together with a PDE inhibitor, IBMX (0.5 μM), had additive effects on rabbit aorta and CCSM. Additionally, KMUP‐2 (100 μM) also affected cGMP metabolism, due to its inhibiting activity on PDE in human platelets. KMUP‐2 (0.1–100 μM) further induced an increase of intracellular cGMP levels in the primary cultured rabbit aortic and CCSM cells. These increases in cGMP content were abolished in the presence of methylene blue (100 μM) and ODQ (10 μM). Obviously, the relaxant effects of KMUP‐2 on rabbit isolated tissues are more sensitive in CCSM than in aorta. Moreover, KMUP‐2 also stimulated NO/sGC/cGMP pathway and subsequent elevation of cGMP by blockade of PDE and enhanced opening of K⁺ channels in rabbit aorta and CCSM. KMUP‐2 (0.2, 0.4, 0.6 mg/kg), similar to KMUP‐1 and sildenafil, caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose‐dependent manner. It is concluded that both the increases of cGMP and the opening activity of K⁺ channels play prominent roles in KMUP‐2‐induced aortic smooth muscle and CCSM relaxation and increases of ICP in rabbits. Drug Dev. Res. 55:162–172, 2002. © 2002 Wiley‐Liss, Inc.     

Honokiol attenuates vascular contraction through the inhibition of the RhoA/Rho-kinase signalling pathway in rat aortic rings

Objectives Honokiol is a small‐molecule polyphenol isolated from the species Magnolia obovata. We hypothesized that honokiol attenuated vascular contractions through the inhibition of the RhoA/Rho‐kinase signalling pathway. Methods Rat aortic rings were denuded of endothelium, mounted in organ baths, and subjected to contraction or relaxation. Phosphorylation of 20 kDa myosin light chains (MLC₂₀), myosin phosphatase targeting subunit 1 (MYPT1) and protein kinase C (PKC)‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase (MLCP) of 17 kDa (CPI17) were examined by immunoblot. We also measured the amount of guanosine triphosphate RhoA as a marker for RhoA activation. Key findings Pretreatment with honokiol dose‐dependently inhibited the concentration–response curves in response to sodium fluoride (NaF) or thromboxane A₂ agonist U46619. Honokiol decreased the phosphorylation levels of MLC₂₀, MYPT1_Thr855 and CPI17_Thr38 as well as the activation of RhoA induced by 8.0 mm NaF or 30 nm U46619. Conclusions These results demonstrated that honokiol attenuated vascular contraction through the inhibition of the RhoA/Rho‐kinase signalling pathway.     

Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

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Inhibitory effects of sertraline in rat isolated perfused kidneys and in isolated ring preparations of rat arteries

Objectives Sertraline is often prescribed to patients suffering with end stage renal disease, but its action on kidney has not been investigated. We aimed to investigate the pharmacological action of sertraline on rat kidney with emphasis on the underlying mechanisms involved in the vascular actions of the drug. Methods The effects of sertraline were evaluated in rat isolated perfused kidneys and on ring preparations of mesenteric or segmental rat renal artery. Key findings In kidneys, sertraline prevented the effects of phenylephrine on perfusion pressure, glomerular filtration rate, urinary flow and renal vascular resistance. In mesenteric rings sertraline inhibited phenylephrine‐induced contractions with potency 30‐times lower than verapamil. Sertraline reversed sustained contractions induced by phenylephrine or 60 mm K⁺ within a similar concentration range. In segmental isolated rings, sertraline also reversed contractions induced by phenylephrine or 60 mm K⁺ with the same concentration range, but with higher potency compared with mesenteric preparations. Under Ca²⁺‐free conditions, sertraline did not change the intracellularly‐mediated phasic contractions induced by phenylephrine or caffeine. Sertraline was ineffective against contractions induced by extracellular Ca²⁺ restoration after thapsigargin treatment and Ca²⁺ store depletion with phenylephrine. Conversely, sertraline decreased the contractions induced by Ca²⁺ addition in tissues under high K⁺ solution or phenylephrine plus verapamil. Conclusions In rat isolated kidneys and in rat ring preparations of mesenteric or renal vessels, sertraline had antispasmodic effects that appeared to be caused by a direct action on vascular smooth muscle cells. Its actions were ineffective against Ca²⁺‐releasing intracellular pathways, but appeared to interfere with sarcolemmal Ca²⁺ influx with reduced permeability of both receptor‐ and voltage‐gated Ca²⁺ channels.     

Interference of pentobarbitone with the contraction of vascular smooth muscle in goat middle cerebral artery

Pentobarbitone (10^?5 to 10^?3 M) decreased the basal tone of vascular smooth muscle of goat middle cerebral artery in a dose-dependent manner as well as relaxing established contractions induced by noradrenaline (NA) (10^?5 M), 5-hydroxytryptamine (5-HT) (10^?5 M) and KCl(120 mM). Preincubations with pentobarbitone reduced the contractions evoked by these three agents in a dose-dependent way. It also decreased Ca²⁺-induced contractile responses in K⁺-depolarized arteries and 5-HT-Ca²⁺ and NA-Ca²⁺ contractions dose-dependently. Contractions induced by K⁺ were more sensitive to the depressant actions of the drug than those produced by NA and 5-HT. The small contractions evoked by K⁺ and 5-HT in Ca²⁺-free medium were also reduced in its presence. The antagonism Ca²⁺-pentobarbitone was insurmountable. These results suggest that the drug interferes with Ca²⁺ entry and Ca²⁺ release from cell stores, and therefore with the smooth muscle contractions.     

Influence of Osmolality of Solutions Used for K+ Contraction on Relaxant Responses to Pinacidil,Verapamil, Theophylline and Terbutaline in Isolated Airway Smooth Muscle

Abstract: Concentration-relaxation profiles for pinacidil, verapamil, terbutaline and theophylline were studied in guinea-pig trachealis contracted by two commonly applied techniques for K⁺ depolarization. All drugs were much less effective on contractions induced by hyperosmolar 124 mM K⁺ solution (added KCl) than on contractions elicited by an isoosmolar 124 mM K⁺ Krebs solution (substituted KCl). The maximal relaxant responses were (isoosmolar K⁺/hyperosmolar K⁺): pinacidil 100%/40%, verapamil 100%/60%, theophylline 100%/0%, terbutaline 50%/0%. Addition of mannitol to establish the same hyperosmolarity as with 124 mM KCl also produced contraction of guinea-pig trachealis. Concentration-relaxation curves for the drugs on mannitol-induced contractions had close resemblance to those obtained in hyperosmolar 124 mM K⁺ solution. When contraction was elicited by 30 mM K⁺, pinacidil showed seven times higher relaxant potency in hyperosmolar compared to isoosmolar solution, whereas the relaxant responses to verapamil, theophylline and terbutaline were not influenced by osmolality. When K⁺ depolarization is used as a tool for evaluation of drug action in airway smooth muscle, the two different techniques produce dissimilar results. The influence of hyperosmolarity pec se appears to be an important and unwanted feature when K⁺ depolarization is produced by addition of KCl.     

Cholinergic neuromuscular transmission mediated by interstitial cells of Cajal in the myenteric layer in mouse ileal longitudinal smooth muscles

To elucidate the roles played by the interstitial cells of Cajal in the myenteric layer (ICC-MY) in cholinergic neuromuscular transmission, we recorded mechanical and electrical activities in response to electrical field stimulation (EFS) of the ileal longitudinal muscle strips from WBB6F1-W/W^V (W/W^V) mutant mice, that lacked ICC-MY and compared with those in WBB6F1-+/+ (+/+) control mice. In +/+ muscle strips, EFS induced phasic contractions, which were abolished or strongly attenuated by atropine or tetrodotoxin. In W/W^V preparations, EFS induced similar phasic contractions, but the cholinergic component was smaller than that in +/+ strips. This was despite of the fact that the contractions because of exogenous applications of carbachol and high K⁺ solution in W/W^V strips were comparable to or rather greater than those in the +/+ preparations. EFS induced atropine-sensitive excitatory junction potentials (EJPs) in the +/+ longitudinal smooth muscle cells but not in W/W^V cells. In the presence of eserine, EFS induced atropine-sensitive EJPs in W/W^V cells. These results suggest that ICC-MY mediate the cholinergic neuromuscular transmission in mouse ileal longitudinal smooth muscles. In addition, the other pathway in which ICC-MY are not involved can operate concomitantly.     

Sphingosine-1-phosphate augments agonist-mediated contraction in the bronchial smooth muscles of mice

The effects of sphingosine-1-phosphate (S1P) on bronchial smooth muscle (BSM) contractility were investigated in naive mice. S1P had no effect on the basal tone of the isolated BSM tissues. However, in the presence of S1P (10(-6) M), the BSM contractions induced by acetylcholine (ACh) and endothelin-1 (ET-1) were significantly augmented: both the ACh and ET-1 concentration-response curves were significantly shifted to the left. In contrast, the pretreatment with S1P had no effect on the contractions induced by high K(+) depolarization. It is thus possible that S1P augments BSM contraction induced by the activation of G protein-coupled receptors.     

Unique effect of tetrapentylammonium ions on sympathetic transmission in rat vas deferens

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Calcium Antagonistic Properties of the Sesquiterpene T-Cadinol: A Comparison with Nimodipine in the Isolated Rat Aorta

Abstract: (+)-T-Cadinol is a sesquiterpene with smooth muscle relaxing properties. In the isolated rat aorta, T-cadinol relaxed contractions induced by 60 mM K⁺ in a concentration-dependent fashion. The dihydropyridine calcium antagonist nimodipine was approximately 4,000 times more potent than T-cadinol. While both drugs nearly abolished the K⁺-induced contractions, they only partially relaxed contractions induced by phenylephrine. The relaxation induced by T-cadinol and nimodipine in K⁺-contracted aortic rings, was completely reversed by the calcium channel activator Bay K8644. In aortic preparations partially depolarized by 20 mM K⁺, Bay K8644 induced a concentration-dependent contraction. Nimodipine shifted the Bay K8644 concentration-response curve to the right in a parallel manner, consistent with a competitive mode of inhibition. T-cadinol at concentrations less than 10^?3.5 M also produced a right-ward shift of the Bay K8644 concentration-response curve with a maintained maximum response. However, the highest T-cadinol concentration used (10^?3.5 M) significantly reduced the maximum response. In conclusion, although T-cadinol and nimodipine display marked structural differences, their pharmacological profiles of action have several features in common, suggesting that T-cadinol is a calcium antagonist, possibly interacting with the dihydropyridine binding sites on the calcium channels.     

Impairment of α1‐adrenoceptor‐mediated contractile activity in caudal arterial smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. In the present study, we compared the responsiveness of de‐endothelialized caudal artery smooth muscle strips, isolated from Type 2 diabetic Goto‐Kakizaki (GK) and normal Wistar rats, to α₁‐adrenoceptor stimulation (cirazoline) and membrane depolarization (K⁺). 2. The contractile and myosin 20 kDa light chain (LC₂₀) phosphorylation responses to 0.3 μmol/L cirazoline of caudal artery strips isolated from 12‐week‐old GK rats were significantly reduced compared with those of age‐matched Wistar rats, whereas the contractile and LC₂₀ phosphorylation responses to 60 mmol/L K⁺ were unaltered. 3. Stimulation of fura 2‐AM‐loaded strips from GK rats with 0.3 μmol/L cirazoline induced a significantly smaller rise in [Ca²⁺]_i (by ～20%) compared with that in strips from Wistar rats, whereas comparable Ca²⁺ transients were evoked by K⁺ in both. 4. Using quantitative polymerase chain reaction, no significant differences were detected in the mRNA expression of α_1A‐, α_1B‐ and α_1D‐adrenoceptor subtypes between GK and Wistar rats. 5. Cirazoline (1 μmol/L)‐ and caffeine (20 mmol/L)‐induced contractions in the absence of extracellular Ca²⁺ were unaltered in GK rats, suggesting that the release of Ca²⁺ from the sarcoplasmic reticulum in response to cirazoline does not differ between GK and Wistar rats. 6. The results of the present study suggest that Ca²⁺ entry from the extracellular space via α₁‐adrenoceptor‐activated, Ca²⁺‐permeable channels, but not via membrane depolarization and voltage‐gated L‐type Ca²⁺ channels, is impaired in caudal artery smooth muscle of GK rats.     

TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation

BACKGROUND AND PURPOSE

TASK1 (K_2P3.1) two-pore-domain K⁺ channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1.

EXPERIMENTAL APPROACH

Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes.

KEY RESULTS

ET-1 inhibited TASK1-mediated I_KN currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET_A (IC₅₀= 0.08 nM) and ET_B (IC₅₀= 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser³³⁶ and Ser³⁹³. Mutation of Ser³⁹³ rendered TASK1 channels insensitive to ET_A- or ET_B-mediated current inhibition. In contrast, removal of Ser³³⁶ selectively attenuated ET_A-dependent TASK1 regulation without affecting the ET_B pathway.

CONCLUSIONS AND IMPLICATIONS

ET-1 regulated vascular TASK1 currents through ET_A and ET_B receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.     

Effect of potassium channel opener pinacidil on the contractions elicited electrically or by noradrenaline in the human radial artery

In order to discover an agent that can prevent spasm of the human radial artery, the aim of our study was to evaluate the effect of the K⁺ channel opener, pinacidil, on contractions in the radial artery. Contractions of the radial artery were evoked by exogenously applied noradrenaline or by electrical field stimulation (EFS, 20 Hz, neurogenic). Pinacidil induced concentration-dependent inhibition of both EFS- and noradrenaline-evoked contractions of the radial artery. Glibenclamide, a selective blocker of ATP-sensitive K⁺ channels (Kir6.x containing subunit) antagonized in the same manner the pinacidil-induced inhibition of neurogenic contractions and contractions evoked by exogenous noradrenaline. The inhibition of pinacidil relaxation by tetraethylammonium (TEA), a blocker of Ca-sensitive K⁺ (K_Ca) channels, was more pronounced in EFS-contracted preparations. A blocker of voltage-sensitive K⁺ (K_V) channels, 4-aminopyridine (4-AP), inhibited pinacidil relaxation only in EFS-contracted preparations. In order to test the presence of different K⁺ channels, immunohistochemistry of K⁺ channels expression in the radial artery was performed. The vascular wall of the human radial artery showed variable positivity with the following applied antibodies: Kv1.2, Kv1.3, Kir6.1, and K_Ca1.1. The antibodies against Kv1.6, Kv2.1, and Kir6.2 channel subunits were completely negative. These results suggest that the inhibitory effect of pinacidil on contractions of the human radial artery might be postsynaptic and associated with opening of smooth muscle Kir6.1-containing K_ATP channels. TEA- and 4-AP-sensitive K⁺ channels may also contribute to pinacidil effect in the human radial artery.     

Analysis of Adenosine Vascular Effect in Isolated Rat Aorta: Possible Role of Na+/K+‐ATPase

Abstract: The present experiments were undertaken in order to examine the effect of adenosine in isolated rat aorta, to investigate the possible role of intact endothelium and endothelial relaxing factors in this action and to determine which population of adenosine receptors is involved in rat aorta response to adenosine. Adenosine (0.1–300 μM) produced concentration‐dependent (intact rings: pD₂=4.39±0.09) and endothelium‐independent (denuded rings: pD₂=4.52±0.12) relaxation of isolated rat aorta. In the presence of high concentration of K⁺ (100 mM) adenosine‐evoked relaxation was significantly reduced (maximal relaxation in denuded rings: control – 92.1±9.8 versus K⁺– 54.4±5.0). Similar results were obtained after incubation of ouabain (100 μM) or glibenclamide (1 μM). In K⁺‐free solution, K⁺ (1–10 mM)‐induced rat aorta relaxant response was significantly inhibited by ouabain (100 μM). Application of indomethacin (10 μM), N^G‐nitro‐L‐arginine (10 μM) or tetraethylammonium (500 μM) did not alter the adenosine‐elicited effect in rat aorta. 8‐(3‐Chlorostyril)‐caffeine (0.3–3 μM), a selective A_2A‐receptor antagonist, significantly reduced adenosine‐induced relaxation of rat aorta in a concentration‐dependent manner (pK_B=6.57). Conversely, 1,3‐dipropyl‐8‐cyclopentylxanthine (10 nM), an A₁‐receptor antagonist, did not affect adenosine‐evoked dilatation. These results indicate that in isolated rat aorta, adenosine produces endothelium‐independent relaxation, which is most probably dependent upon activation of smooth muscle Na⁺/K⁺‐ATPase, and opening of ATP‐sensitive K⁺ channels, to a smaller extent. According to receptor analysis, vasorelaxant action of adenosine in rat aorta is partly induced by activation of smooth muscle adenosine A_2A receptors.