Hydrogen peroxide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

This study was designed to determine whether or not endothelium-dependent hyperpolarizations evoked by acetylcholine in the isolated guinea-pig carotid artery involve hydrogen peroxide. Membrane potential was recorded in the vascular smooth muscle cells of that artery. Under control conditions, acetylcholine induced endothelium-dependent hyperpolarization of the vascular smooth muscle cells which was not affected by the presence of catalase, superoxide dismutase or their combination. Neither the superoxide dismutase mimetic, tiron nor the thiol-reducing agent N-acetyl-l-cysteine modified the hyperpolarization evoked by 0.1 μM acetylcholine but each produced a partial and significant inhibition of the hyperpolarization induced by 1 μM acetylcholine. Neither 10 nor 100 μM hydrogen peroxide influenced the resting membrane potential of the smooth muscle cells and the higher concentration did not significantly influence the hyperpolarization elicited by acetylcholine. These data indicate that, in the guinea-pig isolated carotid artery, hydrogen peroxide is unlikely to contribute to the endothelium-dependent hyperpolarization evoked by acetylcholine.     

C-type natriuretic peptide and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

BACKGROUND AND PURPOSE: C-type natriuretic peptide (CNP) has been proposed to make a fundamental contribution in arterial endothelium-dependent hyperpolarization to acetylcholine. The present study was designed to address this hypothesis in the guinea-pig carotid artery. EXPERIMENTAL APPROACH: The membrane potential of vascular smooth muscle cells was recorded in isolated arteries with intracellular microelectrodes. KEY RESULTS: Acetylcholine induced endothelium-dependent hyperpolarizations in the presence or absence of N (G)-nitro-L-arginine, indomethacin and/or thiorphan, inhibitors of NO-synthases, cyclooxygenases or neutral endopeptidase, respectively. Acetycholine hyperpolarized smooth muscle cells in resting arteries and produced repolarizations in phenylephrine-stimulated arteries. CNP produced hyperpolarizations with variable amplitude. They were observed only in the presence of inhibitors of NO-synthases and cyclooxygenases and were endothelium-independent, maintained in phenylephrine-depolarized carotid arteries, and not affected by the additional presence of thiorphan. In arteries with endothelium, the hyperpolarizations produced by CNP were always significantly smaller than those induced by acetylcholine. Upon repeated administration, a significant tachyphylaxis of the hyperpolarizing effect of CNP was observed, while consecutive administration of acetycholine produced sustained responses. The hyperpolarizations evoked by acetylcholine were abolished by the combination of apamin plus charybdotoxin, but unaffected by glibenclamide or tertiapin. In contrast, CNP-induced hyperpolarizations were abolished by glibenclamide and unaffected by the combination of apamin plus charybdotoxin. CONCLUSIONS AND IMPLICATIONS: In the isolated carotid artery of the guinea-pig, CNP activates K(ATP) and is a weak hyperpolarizing agent. In this artery, the contribution of CNP to EDHF-mediated responses is unlikely.     

Role of endothelial cell hyperpolarization in EDHF-mediated responses in the guinea-pig carotid artery

1. Experiments were performed to identify the potassium channels involved in the acetylcholine-induced endothelium-dependent hyperpolarization of the guinea-pig internal carotid artery. Smooth muscle and endothelial cell membrane potentials were recorded in isolated arteries with intracellular microelectrodes. Potassium currents were recorded in freshly-dissociated smooth muscle cells using patch clamp techniques. 2. In single myocytes, iberiotoxin (0.1 microM)-, charybdotoxin (0.1 microM)-, apamin (0.5 microM)- and 4-aminopyridine (5 mM)-sensitive potassium currents were identified indicating the presence of large- and small-conductance calcium-sensitive potassium channels (BK(Ca) and SK(Ca)) as well as voltage-dependent potassium channels (K(V)). Charybdotoxin and iberiotoxin inhibited the same population of BK(Ca) but a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected. 4-aminopyridine (0. 1 - 25 mM) induced a concentration-dependent inhibition of K(V) without affecting the iberiotoxin- or the apamin-sensitive currents. 3. In isolated arteries, both the endothelium-dependent hyperpolarization of smooth muscle and the hyperpolarization of endothelial cells induced by acetylcholine or by substance P were inhibited by 5 mM 4-aminopyridine. 4. These results indicate that in the vascular smooth muscle cells of the guinea-pig carotid artery, a conductance specifically sensitive to the combination of charybdotoxin plus apamin could not be detected, comforting the hypothesis that the combination of these two toxins should act on the endothelial cells. Furthermore, the inhibition by 4-aminopyridine of both smooth muscle and endothelial hyperpolarizations, suggests that in order to observe an endothelium-dependent hyperpolarization of the vascular smooth muscle cells, the activation of endothelial potassium channels is likely to be required.     

Epoxyeicosatrienoic acids,potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery

1. Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium.
2. In the presence of N^ω-nitro-L-arginine (L-NOARG, 100 μM) and indomethacin (5 μM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 μM) evoked an endothelium-dependent hyperpolarization which averaged −16.4 mV starting from a resting membrane potential of −56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential.
3. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 μM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 μM) to −12.8±1.2 mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4 mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7 mV, n=8).
4. 17-ODYA (10 μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (−10.9 mV).
5. Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization.
6. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 μM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery.
7. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P₄₅₀ pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BK_Ca channels.

     

Cyclic GMP-independent relaxation and hyperpolarization with acetylcholine in guinea-pig coronary artery.

1. The effects of acetylcholine (ACh) on membrane potential, relaxation and cyclic GMP levels were compared to the NO donor L-nitrosocysteine (Cys-NO) in segments of guinea-pig coronary artery. 2. ACh and Cys-NO produced concentration-dependent relaxations of muscles contracted with the H1 receptor agonist, 2-(2-aminoethyl)pyridine (AEP, 0.35 mM). The relaxation to ACh was unchanged in the presence of NG-monomethyl-L-arginine (L-NMMA; 350 microM) or indomethacin (3 microM). 3. Oxyhaemoglobin (HbO; 20 microM) alone or in combination with L-NMMA increased the EC50 for ACh-induced relaxation whereas relaxation with Cys-NO was almost completely abolished with HbO. 4. Scorpion venom (SV; 8.7 micrograms ml-1) increased the EC50 for relaxation with ACh but not Cys-NO. Combined L-NMMA, HbO and SV produced nearly complete abolition of ACh-induced relaxations. 5. Basal cyclic GMP levels (i.e., 20 pmol mg-1 protein) were significantly increased following addition of either ACh (190 pmol mg-1 protein) or Cys-NO (240 pmol mg-1 protein). L-NMMA significantly reduced the rise of cyclic GMP with ACh but not Cys-NO. In contrast, SV did not significantly reduce the rise in cyclic GMP produced with ACh. In the combined presence of L-NMMA and HbO neither ACh nor Cys-NO produced a significant increase in cyclic GMP levels. 6. ACh gave rise to significantly greater membrane hyperpolarization than Cys-NO both in the presence and absence of AEP. Combined L-NMMA and HbO did not reduce the amplitude of hyperpolarization with ACh.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of endothelium-dependent hyperpolarization by endothelial prostanoids in guinea-pig coronary artery

1. In smooth muscle of the circumflex coronary artery of guinea-pig, acetylcholine (ACh, 10(-6) M) produced an endothelium-dependent hyperpolarization consisting of two components. An initial component that occurs in the presence of ACh and a slow component that developed after ACh had been withdrawn. Each component of the hyperpolarization was accompanied by an increase in membrane conductance. 2. Indomethacin (5 x 10(-6) M) or diclofenac (10(-6) M), both inhibitors of cyclooxygenase, abolished only the slow hyperpolarization. The initial hyperpolarization was not inhibited by diclofenac nor by nitroarginine, an inhibitor of nitric oxide synthase. 3. Both components of the ACh-induced hyperpolarization were abolished in the presence of atropine (10(-6) M) or high-K solution ([K+]0 = 29.4 mM). 4. The interval between ACh-stimulation required to generate an initial hyperpolarization of reproducible amplitude was 20 min or greater, but it was reduced to less than 5 min after inhibiting cyclooxygenase activity. Conditioning stimulation of the artery with substance P (10(-7) M) also caused a long duration (about 20 min) inhibition of the ACh-response. 5. The amplitude of the hyperpolarization generated by Y-26763, a K+-channel opener, was reproducible within 10 min after withdrawal of ACh. 6. Exogenously applied prostacyclin (PGI2) hyperpolarized the membrane and reduced membrane resistance in concentrations over 2.8 x 10(-9)M. 7. At concentrations below threshold for hyperpolarization and when no alteration of membrane resistance occurred, PGI2 inhibited the initial component of the ACh-induced hyperpolarization. 8. It is concluded that endothelial prostanoids, possibly PGI2, have an inhibitory action on the release of endothelium-derived hyperpolarizing factor.     

Endothelium-dependent relaxation and hyperpolarization in guinea-pig coronary artery: role of epoxyeicosatrienoic acid

1. Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 μM N^ω-nitro-L-arginine and 10 μM indomethacin.
2. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H₁-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 μM).
3. The cytochrome P450 inhibitors proadifen (10 μM) and clotrimazole (10 μM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 μM) and 7-ethoxyresorufin (10 μM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 μM) and LEM (1 μM)-induced hyperpolarization.
4. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 μM), glibenclamide (10 μM) and BaCl₂ (50 μM) had no significant effect on responses to ACh, AA and 11,12-EET.
5. IBTX (100 nM) significantly reduced both 11,12-EET (33 μM) and AA (30 μM) hyperpolarization without affecting the ACh (1 μM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations.
6. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

     

Isoprenaline- and noradrenaline-induced hyperpolarization of guinea-pig liver cells

1 Effects of pretreatment with isoprenaline (Isop) or noradrenaline (NA) and various ionic environments on the NA-induced or Isop-induced hyperpolarization of guinea-pig liver cells were investigated by means of a microelectrode technique.

2 NA (5.9 × 10^-6 M) decreased the membrane resistance, and hyperpolarized the membrane with or without generation of an initial transient small depolarization. The NA-induced initial depolarization was not dependent on the membrane potential and was increased by Isop (4.0 × 10^-6 M) or glucagon (10^-7 M).

3 In Ca-free solution, the NA-induced hyperpolarization became transient and a continuous depolarization followed in the presence of NA. Repetitive application of NA resulted in a complete disappearance of the NA-induced hyperpolarization and was replaced by a slowly developing depolarization with or without generation of the initial transient depolarization. In excess [Ca]_o, the NA or Isop-induced hyperpolarization was increased.

4 Both Isop and glucagon hyperpolarized the membrane and decreased the membrane resistance, to various degrees. Repetitive application of Isop or glucagon resulted in the disappearance of both Isop and glucagon-induced hyperpolarizations. Pretreatment with NA not only resulted in a recovery of both Isop and glucagon-induced hyperpolarizations, but also extensively enhanced the hyperpolarization.

5 After pretreatment with Isop, the NA-induced hyperpolarization was decreased in amplitude and duration and was followed by a slowly developing depolarization. After repetitive application of Isop, NA produced only depolarization of the membrane, and in these conditions, Isop, glucagon or ATP also depolarized the membrane. These depolarizations were reversed to hyperpolarizations by pretreatment with excess [Ca]_o.

6 After treatment with Na-deficient solution, NA depolarized the membrane and decreased the membrane resistance. Excess [Ca]_o restored the NA-induced membrane response from one of depolarization to one of hyperpolarization.

7 In the presence of tetraethylammonium 10mM, the NA-induced hyperpolarization became transient or ceased and depolarization occurred with a reduction in the membrane resistance.

8 It is postulated that both NA and Isop increase the free [Ca]_i by releasing bound Ca from storage sites and consequently an increase in K conductance follows. NA but not Isop promotes Ca-influx which replenishes the storage site. In Ca-depleted conditions, NA does not elevate the free [Ca]_i to a threshold concentration required for hyperpolarization, probably because NA induces a small release of Ca from storage sites.

     

Inhibitors of the cytochrome P450-mono-oxygenase and endothelium-dependent hyperpolarizations in the guinea-pig isolated carotid artery.

1. Transmembrane potentials were recorded from isolated carotid arteries of the guinea-pig superfused with modified Krebs-Ringer bicarbonate solution. Smooth muscle cells were impaled with sharp intracellular microelectrodes. 2. Acetylcholine (1 microM) induced an endothelium-dependent hyperpolarization (14.3 +/- 2.8 mV, n = 6) which was not affected (15.1 +/- 1.1 mV, n = 35) by inhibitors of cyclo-oxygenase (indomethacin, 5 microM) and nitric oxide synthase (N omega nitro-L-arginine: L-NOARG, 100 microM). 3. The hyperpolarization produced by acetylcholine was abolished in the presence of elevated potassium (35 mM) in the superfusing physiological saline solution. 4. The acetylcholine-induced hyperpolarization was not affected by the inhibitors of cytochrome P450 mono-oxygenases, SKF525a (10 and 100 microM, 13.9 +/ 2.2 and 15.3 +/- 4.6 mV), metyrapone (100 microM, 13.1 +/- 1.9 mV), clotrimazole (100 microM, 13.5 +/- 2.7 mV), 17-octadecynoic acid (5 microM, 16.5 +/- 1.9 mV), methoxsalen (10 microM, 15.3 +/- 1.6 mV), the inhibitor of phospholipase A2 quinacrine (10 microM 12.8 +/- 2.5 mV) and the non specific lipoxygenases/cyclo-oxygenases/cytochrome P450 inhibitor, eicosatetraynoic acid (50 microM, 15.0 +/- 2.2 mV). However, the muscarinic antagonist, atropine (100 nM), abolished the hyperpolarization. 5. These results suggest that in guinea-pig carotid artery, the metabolism of arachidonic acid, either through cyclo-oxygenase, lipoxygenase or cytochrome p450 mono-oxygenase, is not involved in acetylcholine-induced endothelium-dependent hyperpolarizations.     

Differential effects of prostacyclin and iloprost in the isolated carotid artery of the guinea-pig

The effects on membrane potential of prostacyclin and iloprost were compared in smooth muscle cells of the guinea pig carotid artery. Both prostacyclin and iloprost induced hyperpolarization of the smooth muscle cells. In the presence of (3R)-3-(4-fluorophenyl-sulfonamido)-1,2,3,4-tetrahydro-9-carbazolepropanoic acid (Bay U3405), an antagonist of TP receptors, the response to iloprost was unaffected while that to prostacyclin was increased. Iloprost-induced hyperpolarizations were abolished by glibenclamide while those to prostacyclin were either not affected, or converted to either depolarization or to rhythmic electrical activity. The latter effects of prostacyclin were abolished by Bay U3405. After removal of the endothelium, iloprost and prostacyclin produced hyperpolarizations similar to those observed in control blood vessels. However, in the presence of glibenclamide, prostacyclin produced only depolarizations inhibited by Bay U3405. These results suggest that iloprost activates IP receptors and K(ATP) channels in smooth muscle. In contrast, prostacyclin produces additional endothelium-dependent and -independent effects via activation of TP receptors.     

Endothelium-dependent hyperpolarization to acetylcholine in carotid artery of guinea pig: role of lipoxygenase

This study was designed to determine whether lipoxygenase-dependent metabolites of arachidonic acid are involved in the endothelium-dependent hyperpolarization of the guinea pig carotid artery. The membrane potential of vascular smooth muscle cells was measured with intracellular microelectrodes and potassium channels were studied on freshly isolated cells with the patch-clamp technique. Acetylcholine-induced hyperpolarizations were not affected by arachidonyl trifluoromethyl ketone (AACOCF3), quinacrine (phospholipase A inhibitors), or eicosatetraenoic acid (nonspecific inhibitor of lipoxygenase, cytochrome P450, and cyclooxygenase). In contrast, cinnamyl-3,4 dihydroxy-alpha-cyanocinnamate (CDC) and AA861 (lipoxygenase inhibitors) as well as 1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C inhibitor) produced a significant inhibition of the hyperpolarization. An opener of intermediate conductance calcium-activated potassium channels, 1-ethyl-2-benzamidazolinone (1-EBIO), induced a hyperpolarization that was unaffected by AACOCF3, CDC, AA861, or U-73122 but was inhibited by charybdotoxin. (+/-)12-hydroxy-eicosatetraenoic acid (12-HETE) and 12(S)-hydroperoxy-eicosatetraenoic acid (12(S)-HpETE) did not induce any significant changes in membrane potential. CDC inhibited the voltage-gated potassium current and increased the large conductance calcium-activated potassium current whereas AA861 inhibited both potassium currents. These results confirm that, in the isolated carotid artery of the guinea pig, stimulation of endothelial muscarinic receptors involves phospholipase C activation and indicate that the activation of phospholipase A2 and the release of lipoxygenase metabolites is unlikely to explain endothelium-dependent hyperpolarization.     

Membrane hyperpolarization, cyclic nucleotide levels and relaxation in the guinea-pig internal anal sphincter.

1. Changes in membrane potential (measured with an intracellular microelectrode) and in cyclic nucleotide (adenosine 3':5'-cyclic monophosphate, cyclic AMP and guanosine 3':5'-cyclic monophosphate, cyclic GMP) levels (measured by radioimmunoassay) in response to inhibitory non-adrenergic non-cholinergic (NANC) field stimulation and drugs were investigated in the guinea-pig internal and anal sphincter (gpIAS) in the presence of phentolamine and atropine (each 10(-6) M). 2. Inhibitory NANC nerve stimulation (single pulse, 5 pulses at 5, 10 and 20 Hz, 0.5 ms supramaximal voltage) and adenosine triphosphate (ATP, 10(-7)-10(-3) M) inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of inhibitory nerve stimulation were blocked by tetrodotoxin (TTX, 10(-6) M) and, with those of ATP, were blocked by apamin (5 x 10(-6) M). 3. Isoprenaline (10(-9)-10(-4) M), cromakalim (10(-9)-10(-5) M), sodium nitroprusside (NaNP 10(-5) M), M&B 22948 (10(-4) M) and 8-bromocyclic GMP (8-Br-cyclic GMP, 10(-4) M) also inhibited spike discharge, hyperpolarized the membrane and relaxed the sphincter. The effects of isoprenaline were blocked by propranolol (10(-6) M). However, forskolin (10(-9)-10(-7) M), M&B 22948 (10(-9)-10(-5) M) and lower concentrations of NaNP (10(-9)-10(-6) M) relaxed the sphincter without affecting the membrane potential. 4. The characteristics of the membrane potential changes in response to different inhibitory stimuli in the gpIAS differed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological distinction of the hyperpolarization response to caffeine and acetylcholine in guinea-pig coronary endothelial cells.

Membrane potential changes in endothelial cells in response to caffeine and acetylcholine (ACh) were recorded with microelectrodes from an intact endothelium preparation from the guinea-pig coronary artery. Caffeine induced a transient hyperpolarization of the membrane in a concentration-dependent manner. The hyperpolarization was inhibited by removal of Ca2+ from the bathing medium and by ryanodine (20 microM). It was not affected by 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester hydrochloride (TMB-8, 10 microM) or neomycin (5 mM). ACh induced a sustained hyperpolarization in endothelial cells. At concentrations that caused no significant effects on the caffeine response, TMB-8 and neomycin inhibited hyperpolarization induced by ACh. Ryanodine did not inhibit the response to ACh. The ACh-induced hyperpolarization was also inhibited by caffeine in a concentration-dependent manner. Results from the present study suggest that hyperpolarizations induced by caffeine and ACh are mediated by separate Ca2+ pools.     

Calmodulin antagonists inhibit endothelium-dependent hyperpolarization in the canine coronary artery.

1. The effects of the calmodulin antagonists, calmidazolium and fendiline were investigated on endothelium-dependent hyperpolarization in the canine coronary artery. The membrane potential of vascular smooth muscle cells was measured with the microelectrode technique. 2. Smooth muscle cells of the canine coronary artery had a resting membrane potential of -50 mV. Bradykinin and the Ca(2+)-ionophore, A23187, induced concentration- and endothelium-dependent hyperpolarization. The hyperpolarization induced by a supramaximal concentration of bradykinin (10(-6) M) reached approximately 20 mV. 3. Calmidazolium (10(-5) M) and fendiline (10(-4) M) inhibited hyperpolarization induced by bradykinin and A23187. By contrast, calmidazolium did not affect the hyperpolarization induced by lemakalim, an opener of ATP-sensitive K(+)-channels. 4. These observations suggest that calmodulin is involved in the generation of endothelium-dependent membrane hyperpolarization of vascular smooth muscle.     

Different effector mechanisms for ATP and adenosine hyperpolarization in the guinea-pig taenia coli

The hyperpolarizations in response to ATP and adenosine in the guinea-pig taenia coli, measured by the sucrose-gap technique at room temperature, were compared in solutions of modified ionic composition. ATP hyperpolarization was increased in low chloride and in low potassium, but was reduced to 12% of control in calcium-free solution on second application of the agonist. The response to adenosine, however, was decreased in low chloride, unchanged in low potassium and was 45% of control in calcium-free solution. The different mechanisms for ATP and adenosine hyperpolarization provide evidence for the presence of separate receptors.     

Thapsigargin- and cyclopiazonic acid-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The present study was designed to determine whether putative, selective inhibitors of the Ca(2+)-pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce endothelium-dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique. 2. In tissues with endothelium, TSG (10(-8)-10(-5) M) caused sustained hyperpolarization in a concentration-dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10(-5) M) also hyperpolarized the smooth muscle membrane, an effect that was endothelium-dependent and long-lasting. 3. The hyperpolarizing responses to these agents were not affected by indomethacin or NG-nitro-L-arginine (L-NOARG). 4. In Ca(2+)-free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response. 5. In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium-dependent relaxations. L-NOARG significantly inhibited the relaxations to these agents, but about 40-60% of the total relaxation was resistant to L-NOARG. The L-NOARG-resistant relaxations were abolished by potassium depolarization. 6. These results indicate that TSG and CPA can cause endothelium-dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium-derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium-dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca(2+)-pump ATPase activity.     

Endothelium-derived factors and k+ channels are involved in the vasorelaxation induced by Sida cordifolia L. in the rat superior mesenteric artery

The vasorelaxantion of the aqueous fraction of the hydroalcoholic extract of the Sida cordifolia leaves (AFSC) was evaluated in this work. In rat superior mesenteric artery, AFSC (3-1000 microg/mL) induced relaxation of phenylephrine-induced contractions. This effect was significantly attenuated after removal of the endothelium, after atropine (1 microM), L-NAME (100 microM), indomethacin (10 microM), high K+ (20 mM), tetraethylammonium (1 microM), a K(Ca) blocker, apamin (1 microM), a SK(Ca) blocker and ChTX (0.1 microM), a BK(Ca) blocker, however, it was not affected after glibenclamide (10 microM), an KATP blocker, and 4-aminopyridine (1 microM), a Kv blocker. ChTX (0.1 microM) was able to induce an additional inhibition of the vasorelaxation induced by AFSC in the presence of L-NAME plus indomethacin. The vasorelaxation induced by AFSC in the presence of L-NAME plus indomethacin plus ChTX was not different from that induced by AFSC in rings without endothelium. In conclusion, the results show that endothelium-derived factors (mainly NO, PGI2) and K+ channels (BK(Ca) and SK(Ca)) play a crucial role in the vasorelaxation induced by AFSC in the rat superior mesenteric artery.     

Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.     

Congenital absence of the internal carotid artery.

A case of a 27 year old male who was found to have the unusual congenital absence of one internal carotid artery is presented. A second case with similar but slightly different X-ray findings due to occlusion of the internal carotid is presented for comparison. Only forty cases of agenesis of the internal carotid artery have been found by us in the literature and this appears to be the first from Africa.     

Trimetazidine inhibits Na+,K(+)-ATPase activity, and overdrive hyperpolarization in guinea-pig ventricular muscles.

The effect of trimetazidine on Na+,K(+)-ATPase activity or the Na+,K+ pump was studied in guinea pig ventricular muscles with the use of biochemical and electrophysiological methods. The effect of trimetazidine on enzyme activity was compared with that in the liver, jejunum and kidney obtained from the same species. Na+,K(+)-ATPase activity in the heart and liver was significantly and concentration dependently decreased by trimetazidine (above 1.5 x 10(-5) M). Even the highest concentration (1.5 x 10(-4) M) of trimetazidine failed to decrease the Na+,K(+)-ATPase activity in the jejunum and kidney. The membrane potential was recorded in the ventricular muscle with a microelectrode. The hyperpolarization which followed 1-min overdrive stimulation (3.3 Hz) was decreased by trimetazidine (1.5 x 10(-4) M), but the depolarization during the stimulation was not affected by this drug. Ouabain, a potent Na+,K+ pump inhibitor, markedly decreased the overdrive hyperpolarization and increased the depolarization during the stimulation (10(-7), 5 x 10(-7), 10(-6) M). Therefore, the effect of trimetazidine and ouabain on the Na+,K+ pump-mediated alteration in the resting potential is different, suggesting that trimetazidine has additional direct membrane effects, e.g. a decrease in K+ conductance. In conclusion, trimetazidine inhibits Na+,K(+)-ATPase activity and thus the Na+,K+ pump in the ventricular muscles but with an inhibitory effect about 300 times less than that of ouabain. Trimetazidine inhibited the Na+,K(+)-ATPase in the liver as well, but not that in jejunum and kidney.