Cerebral vasodilator properties of Danshen and Gegen: a study of their combined efficacy and mechanisms of actions.

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED50, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a KATP channel inhibitor glibenclamide, a non-selective K(+) channel inhibitor tetraethylammonium (TEA), and by a combination of K(+) channel inhibitors (glibenclamide+TEA+iberiotoxin+4-aminopyridine+barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of KATP currents and inhibited Ca(2+) influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl2-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of KATP channels and inhibition of Ca(2+) influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.

The anti-hypertensive effect of Danshen (Salvia miltiorrhiza) and Gegen (Pueraria lobata) formula in rats and its underlying mechanisms of vasorelaxation.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Salviae miltiorrhizae (Danshen) and Radix Puerariae lobatae (Gegen) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating cardiovascular disease. AIMS OF THE STUDY: In the present study, an aqueous extract comprising Danshen and Gegen in the ratio of 7:3 (DG) was investigated for its anti-hypertension in vivo and vasodilative activities ex vivo. MATERIALS AND METHODS: The anti-hypertensive effect of DG extract was investigated in spontaneously hypertensive rat (SHR) by measuring systolic blood pressure (SBP). Oral administration of DG extract was started at age of 6 weeks and 14 weeks for the preventive and therapeutic studies, respectively. Blood pressure was measured by tail-cuff method biweekly for 12 weeks. The ex vivo vasodilative activities of DG extract, its dependency on endothelium and the involvement of nitric oxide, prostacyclin and potassium channels were investigated using isolated rat aorta ring in organ bath. RESULTS: For in vivo study, systolic blood pressure was significantly reduced in DG extract-treated groups (90.2 and 300 mg/kg) as compared with the SHR control in both preventive and therapeutic studies. However, DG extract was unable to suppress or delay the onset of hypertension in the preventive study. For ex vivo study, the results showed that DG extract induced a concentration-dependent relaxation in aorta and persisted response was observed with the removal of endothelium. Besides, pretreatment with a non-selective potassium channel inhibitor tetraethylammonium (TEA) also significantly inhibited DG extract-induced vasodilation. Further investigations on specific potassium channel blockers revealed that ATP-sensitive potassium (K(ATP)) channel inhibitor glibenclamide, inward rectifier potassium (Kir) inhibitor barium chloride and voltage-dependent potassium (K(v)) channel inhibitor 4-aminopyridine, but not BK(Ca) channel inhibitor iberiotoxin, exerted significant inhibition on DG extract-induced vasodilation. CONCLUSIONS: The results of in vivo SHR animal model suggested that DG aqueous extract possessed blood pressure lowering effect on both pre- and post-hypertensive rats, which could be explained by its endothelium-independent vasodilation via the opening of K(ATP), Kir and K(v) channels.

Modulation of Ca(2+)-dependent K(+) currents in mesenteric arterial smooth muscle cells by adenosine.

The effect of adenosine on Ca(2+)-dependent K(+) currents was studied in freshly isolated smooth muscle cells from rat mesenteric artery. Perforated-patch recordings showed that adenosine induced transient outward currents and an overall increase in the averaged currents at higher depolarizing potentials. The changes in current activity induced by adenosine could be blocked by iberiotoxin. The transient outward currents were not dependent directly on external Ca(2+) and could be induced after brief exposure to Ca(2+)-free solutions. In conventional whole-cell recordings, transient outward currents were also induced by adenosine when a low EGTA concentration of 0.1 mM was included in the pipette solution. Adenosine was not effective in inducing increases in outward currents when a higher concentration of 5.0 mM EGTA was used. Ryanodine and thapsigargin were also effective in blocking the effect of adenosine. These observations suggest that adenosine may activate Ca(2+)-dependent K(+) currents by inducing Ca(2+) release from ryanodine-sensitive channels in the sarcoplasmic reticulum of rat mesenteric arterial cells.

Effects of paxilline on K+ channels in rat mesenteric arterial cells.

The effects of paxilline, a mycotoxin, on whole-cell outward currents from freshly isolated cells of the rat mesenteric artery were studied. Paxilline inhibited a component of the outward current that was also sensitive to iberiotoxin. Inhibition could be observed at a concentration of 10 nM and complete inhibition of the iberiotoxin-sensitive current was achieved at 300 nM. The inhibition could be described by a single site of interaction with a Ki of 35.7 nM. Paxilline had no effect on the component of the current that was sensitive to 4-aminopyridine. It is concluded that paxilline is a potent inhibitor of large conductance Ca2+ -activated K+ currents in vascular smooth muscle cells.

Regulation of vascular tone by endothelium-derived hyperpolarizing factor.

1. Endothelium-derived hyperpolarizing factor (EDHF) mediates the nitric oxide (NO)-independent component of the relaxation in rat mesenteric arteries. The relationship between hyperpolarization and vascular tone was studied by simultaneous recording of membrane potential with intracellular microelectrodes and tension in ring segments of rat mesenteric arteries. 2. By depolarizing arteries with high potassium solutions, it was determined that the threshold for contraction is approximately -46 mV. Maximum contraction was attained when the arteries were depolarized to -20 mV. Thus, 1 mV depolarization resulted in an approximate 4% increase in tone. This relationship was not altered in spontaneously hypertensive rats. 3. Noradrenaline (0.3 mumol/L) caused contraction and depolarized arteries by 13 mV. Acetylcholine caused endothelium-dependent relaxation and hyperpolarization up to 14 mV. In the presence of N omega-nitro-L-arginine, the EDHF-mediated relaxation was correlated to hyperpolarization. A hyperpolarization of 1 mV corresponded to a 4.3% decrease of the induced tone. 4. At concentrations (10 mumol/L) causing total relaxation, the maximum hyperpolarization induced by NO was only 7.6 mV. 5. A maximum relaxation of 88% was observed with pinacidil (3 mumol/L), despite a 25 mV hyperpolarization. Relaxations to NO and pinacidil were not correlated with hyperpolarization. At similar levels of hyperpolarization, NO and pinacidil elicited more relaxation than EDHF. 6. These studies show that vascular tone is very sensitive to membrane potential change in the range between -46 and -20 mV in the rat mesenteric artery. The relaxation response to EDHF, unlike that to NO and pinacidil, can be accounted for solely by its effect on the membrane potential.

Effect of K(+)-channel blockers on ACh-induced hyperpolarization and relaxation in mesenteric arteries.

Acetylcholine (ACh) induces endothelium-dependent hyperpolarization in the rat mesenteric artery in the presence of the nitric oxide synthase inhibitor N omega-nitro-L-arginine. We have now studied the effects of K(+)-channel blockers on the hyperpolarization responses to ACh in resting and norepinephrine-contracted rat mesenteric arteries. We also measured tension simultaneously to determine whether the inhibitory effects of these agents on relaxation could be correlated to their effects on hyperpolarization. Glibenclamide had no significant effect on the hyperpolarization or relaxation. Tetraethylammonium (TEA, 5 mM) inhibited the hyperpolarization to ACh significantly to a similar extent in both the resting and norepinephrine-stimulated arteries. Charybdotoxin (100-150 nM) caused only a small but significant inhibition. Apamin (0.3 microM) was the most effective in inhibiting the hyperpolarization in resting arteries. It was less effective in the norepinephrine-contracted arteries. A combination of apamin and charybdotoxin completely abolished the hyperpolarization responses in both conditions. The relaxation to ACh was correlated to hyperpolarization. In all cases, the inhibition of the relaxation by the K(+)-channel blockers could be accounted for by their effects on the hyperpolarization. These results indicate that Ca(2+)-activated K(+)-channels, especially those sensitive to apamin, may be the major ion channels mediating endothelium-dependent hyperpolarization to ACh.

Modulation of endothelium-dependent hyperpolarization and relaxation to acetylcholine in rat mesenteric artery by cytochrome P450 enzyme activity.

Acetylcholine (ACh) induced hyperpolarization and relaxation in rat mesenteric arteries contracted with norepinephrine, as indicated from studies with simultaneous microelectrode and tension recordings. We tested whether the hyperpolarization to ACh was modified by induction and depletion of cytochrome P450 enzymes. Enzyme induction by treating the animals with 3-methylcholanthrene and beta-naphthoflavone for 3 days resulted in a significant increase in the endothelium-dependent hyperpolarization to a maximum of 22.7 +/- 1.0 mV from 13.9 +/- 0.4 mV in arteries from untreated animals. Enzyme depletion by treating the animals with CoCl2 for 2 days resulted in a significant reduction in the maximum hyperpolarization to 9.9 +/- 0.7 mV. When NO synthesis was inhibited by N omega-nitro-L-arginine, the relaxation was correlated to hyperpolarization. The N omega-nitro-L-arginine-resistant responses were significantly inhibited by clotrimazole. The relaxation to ACh was not altered by enzyme induction but was significantly reduced by enzyme depletion. In KCI-contracted arteries, modification of cytochrome P450 enzyme activity had no significant effect on the relaxation to ACh. Similarly, hyperpolarization and relaxation to pinacidil were not significantly affected. These results suggest that the hyperpolarization response to ACh is closely regulated by cytochrome P450-dependent enzymes.

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y.

Neuropeptide Y(NPY) inhibits Ca2+-activated K+ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 microM) had no effect in the absence of intracellular adenosine 5'-triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 microM) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5'-[beta gamma-methylene]-triphosphate (AMP-PCP). NPY inhibited Ca2+-activated K+ channel activity when ATP was replaced by adenosine 5'-O-(3-thiotriphosphate) (ATP [gamma-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 microM), a specific inhibitor of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 microM) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca2+-activated K+ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.

Modulation of Ca(2+)-activated K+ channel activity by tyrosine kinase inhibitors in vascular smooth muscle cell.

The effects of the tyrosine kinase inhibitors genistein, lavendustin A, and tyrphostin A25 on Ca(2+)-activated K+ channel activities in freshly isolated single vascular smooth muscle cells from the rat tail artery were studied by patch clamp recording technique. Genistein (5-50 microM) and lavendustin A (10 microM) increased whole-cell Ca(2+)-activated K+ channel currents. Increase in single channel activities by genistein and lavendustin A was also observed in excised inside-out patches. Diadzein (15 microM), an inactive analogue of genistein, did not alter channel activities. Tyrphostin A25 (10 nM), which had no significant effect on whole-cell currents in concentrations up to 50 microM, increased the open probability of the channels by 841% in inside-out patches. No potentiation of whole-cell and single channel activities by genistein was observed when ATP was omitted from the intracellular solutions. These observations suggest that tyrosine kinase modulates Ca(2+)-activated K+ channel activities in vascular smooth muscle cells.

Ca2+ mobilization by caffeine in single smooth muscle cells of the rat tail artery.

The fluorescent dye fura-2 was used to study the effects of caffeine on cytosolic free Ca2+ level ([Ca2+]i) in freshly isolated single cells from the rat tail artery. Caffeine caused a concentration-dependent transient increase in [Ca2+]i and shortening of the cell. At higher concentrations (> 2 mM), a tonic increase in [Ca2+]i was also observed. The caffeine-induced changes in [Ca2+]i were reproducible with repeated challenges, even though the cells had contracted due to previous exposure to caffeine. Removal of extracellular Ca2+ reduced the resting [Ca2+]i to about half and abolished the tonic Ca2+ increase to caffeine. The transient component was not significantly affected to the first caffeine challenge after Ca2+ removal, but was abolished to the second challenge. Ryanodine (10 microM) significantly inhibited the responses to caffeine while nifedipine and TMB-8-(8-(diethylamino)octyl ester of 3,4,5-trimethoxybenzoic acid) were not effective. Thapsigargin (10-100 microM) induced a sustained increase in [Ca2+]i to 67 nM. The response of caffeine was not affected by thapsigargin. Pretreatment of the cells with noradrenaline (10 microM) abolished subsequent response to caffeine. These results show that Ca2+ responses to caffeine in single cells from the rat tail artery are reproducible with repeated caffeine challenge. Therefore, single cells can be used for comparison studies of the effects of pharmacological agents.

Neuropeptide Y inhibits Ca(2+)-activated K+ channels in vascular smooth muscle cells from the rat tail artery.

The effects of neuropeptide Y (NPY) on the Ca(2+)-activated K+ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single-channel patch-clamp recording techniques. In the presence of nifedipine (1 microM), whole-cell outward currents through Ca(2+)-activated K+ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca(2+)-activated K+ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca(2+)-activated K+ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells.

Membrane rectification in single smooth muscle cells from the rat tail artery.

Membrane rectification to depolarization was studied by voltage recording with patch electrodes in freshly isolated cells from the rat tail artery. Injection of depolarizing currents elicited electrotonic potentials that developed with a single-exponential time course (time constant of 94.8 ms). When the cell was depolarized beyond -30 mV, delayed rectification was observed. A second type of rectification, characterized by oscillations, was observed when the cell was depolarized positive to +30 mV. The threshold of this rectification and the oscillations were sensitive to changes in intracellular Ca2+. Delayed rectification was more sensitive to 4-aminopyridine but more resistant to tetraethylammonium and charybdotoxin than the Ca(2+)-sensitive rectification. A 4-aminopyridine-sensitive outward current (IK,dr) with a threshold of around -30 mV and a second Ca(2+)-sensitive outward current (IK,Ca) activated at around +30 mV were observed from whole-cell voltage clamp recordings. IK,Ca was blocked by tetraethylammonium and charybdotoxin. An 11-pS and a 122-pS channel, having characteristics similar to IK,dr and IK,Ca respectively, were identified from single-channel recordings. These observations showed how membrane depolarization of vascular smooth-muscle cells was regulated by these two populations of K+ channels under various conditions.

Neuropeptide Y potentiates calcium-channel currents in single vascular smooth muscle cells.

The effect of neuropeptide Y (NPY) on Ca(2+)-channel currents in isolated vascular smooth muscle cells was studied with the perforated-patch recording technique. Using Ba2+ (10 mM) as the charge carrier, inward currents sensitive to Cd2+ and nifedipine were potentiated by NPY in a concentration-dependent manner. The threshold concentration for the potentiating effect of NPY was 50 nM and reached a maximum at 150 nM. NPY shifted the steady-state activation curve to less positive membrane potentials by about 6 mV so that the potentiating effect was most prominent near the activation threshold of the current. It had no effect on steady-state inactivation of the current. These results suggest that NPY may potentiate vasoconstriction by promoting calcium entry through L-type voltage-dependent Ca(2+)-channels.

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.

Characterization of acetylcholine-induced membrane hyperpolarization in endothelial cells.

The characteristics of the hyperpolarization response to acetylcholine (ACh) in endothelial cells from the guinea pig coronary artery were studied by microelectrode recording technique. ACh (30 nM to 3 microM) induced membrane hyperpolarization in a dose-dependent manner. The sustenance of the response required the presence of external calcium. The hyperpolarization was not affected by nifedipine (1 microM) but was inhibited by the potassium channel blockers charybdotoxin (10 nM), tetraethylammonium (1 mM), and 4-aminopyridine (0.5 mM). Glibenclamide (10 microM) and apamin (1 microM) were not effective. The inhibitors of endothelium-derived relaxing factor/nitric oxide synthesis N omega-nitro L-arginine (50 microM) and NG-monomethyl L-arginine (30 microM) had no effect on the resting membrane potential or the ACh-induced responses. No hyperpolarization was observed with application of sodium nitroprusside (10 microM) or 8-bromo-cGMP (0.1 microM). Ouabain (10 microM) depolarized the membrane significantly by 5 mV, but the ACh hyperpolarization was not affected. Indomethacin (10 microM) was without effect on the resting membrane potential or the hyperpolarization to ACh. These results show that ACh-induced hyperpolarization is dependent on external calcium and can be inhibited by certain potassium channel blockers. The hyperpolarization response is not mediated by endothelium-derived relaxing factor/nitric oxide, cGMP, a cyclooxygenase product, or stimulation of the Na-K pump.

Calcium activation of hyperpolarization response to acetylcholine in coronary endothelial cells.

Hyperpolarization response to acetylcholine (ACh) in endothelial cells was studied by intracellular recording of an intact endothelium preparation of guinea pig coronary artery. The hyperpolarization requires the presence of extracellular Ca2+ and the response was progressively blocked by MnCl2 or by removal of extracellular Ca2+. The intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8, 10 microM), was also effective in abolishing the hyperpolarization. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+ pump, potentiated the hyperpolarization to ACh but inhibited caffeine-induced hyperpolarization. These findings suggest that ACh-induced hyperpolarization is mediated by an increase in cytosolic Ca2+ due to influx of extracellular Ca2+ and release of Ca2+ from an internal IP3-sensitive store. The rise in cytosolic Ca2+ level was modulated by a caffeine-sensitive pool that is inhibited by cyclopiazonic acid.

Endothelium-independent potentiating effects of neuropeptide Y in the rat tail artery.

The role of the endothelium in the potentiating action of neuropeptide Y (NPY) to contraction induced by KCl, alpha, beta-methylene ATP (mATP), and noradrenaline (NA) was tested on rat tail arteries. Endothelium-intact and denuded ring segments and freshly isolated single smooth muscle cells were used in the study. Contraction responses to KCl and mATP were potentiated by NPY (50 nM) in both intact and denuded arteries. Contraction to NA was potentiated by NPY at 500 nM but not at 50 nM. The potentiation effect of NPY was antagonized by nifedipine. Similarly, the shortening of single smooth muscle cells in response to KCl and mATP was potentiated by NPY (50 nM). The noradrenaline response was potentiated by NPY at 500 nM but not at 50 nM. Our results suggest that the potentiating effect of NPY is more specific to contraction mediated by nifedipine-sensitive calcium channels and is not dependent on the presence of an intact endothelium.

An electrophysiological study on the effect of neuropeptide Y on sympathetic neurotransmission in guinea pig vas deferens.

The effect of neuropeptide Y (NPY) on sympathetic neurotransmission to the guinea pig vas deferens was studied by recording pre- and postsynaptic electrical events with a suction electrode. When applied locally to the recording site by internal perfusion of the suction electrode, NPY inhibited both the nerve action potential (NAP) and the excitatory junction current (EJC). When applied only to the outside bathing solution, NPY had no inhibitory effect on the NAP or the EJC. The site of action of NPY is therefore at the presynaptic nerve terminal. The mode of action of NPY is different from those of B-HT 920, an alpha-2 adrenoceptor agonist, and omega-conotoxin, a neurotoxin that acts specifically on presynaptic calcium channels. Both of these agents inhibited the EJC without any significant effect on the NAP.

Neuropeptide Y potentiates specifically the purinergic component of the neural responses in the guinea pig saphenous artery.

Contraction of the guinea pig saphenous artery induced by stimulation of perivascular nerves consists of an adrenergic and a purinergic component. Neuropeptide Y (NPY) potentiated the neural responses significantly at low stimulation frequencies but not at high frequencies when the contraction was maximal. After blocking the adrenoceptors with phentolamine, significant potentiation of the purinergic component by NPY could be demonstrated. The potentiating effect of NPY was abolished in the presence of nifedipine. After desensitizing the purinoceptors with alpha,beta-methylene-ATP, there was no potentiation of the remaining adrenergic component by NPY. NPY also has no effect on contractions induced by exogenous norepinephrine but potentiates those induced by alpha,beta-methylene-ATP. These results suggest that NPY potentiates neurally induced contraction in the guinea pig saphenous artery by a specific action on the purinergic response.