Role of Endothelial Kinin B1 Receptor on the Membrane Potential of Transgenic Rat Aorta.

The kinin receptors are classically involved in inflammation, pain and sepsis. The effects of the kinin B1 receptor agonist des-Arg9-bradykinin (DBK) and lipopolysaccharide (LPS) were investigated by comparing the membrane potential responses of aortic rings from transgenic rats overexpressing the kinin B1 receptor (B1R) in the endothelium (TGR(Tie2B1)) and Sprague Dawley (SD) rats. No difference in the resting membrane potential in the aorta's smooth muscle from the transgenic and SD rats was observed. The aorta rings from SD rats hyperpolarized only to LPS but not to DBK, whereas the aorta rings from TGR(Tie2B1) responded by the administration of both drugs. DBK and LPS responses were inhibited by the B1 receptor antagonist R715 and by iberiotoxin in both cases. Thapsigargin induced a hyperpolarization in the smooth muscle of SD rats that was not reversed by R715, but was reversed by iberiotoxin and this hyperpolarization was further augmented by DBK administration. These results show that the model of overexpression of vascular B1 receptors in the TGR(Tie2B1) rats represent a good model to study the role of functional B1 receptors in the absence of any pathological stimulus. The data also show that KCa channels are the final mediators of the hyperpolarizing responses to DBK and LPS. In addition, we suggest an interaction between the B1R and TLR4, since the hyperpolarization induced by LPS could be abolished in the presence of R715.

Alpha-2 adrenoceptors are present in rat aorta smooth muscle cells, and their action is mediated by ATP-sensitive K(+) channels.

The role of alpha(2)-adrenoceptors in the response of aorta smooth muscle rings to the alpha(2)-adrenoceptors agonists UK 14,304 and clonidine was studied. Stimulation by 1 - 10 nM UK 14,304 caused dose-dependent relaxant responses in BaCl(2)-contracted endothelium-denuded aorta rings, and hyperpolarization in rings with or without endothelium, which were inhibited by yohimbine and glibenclamide, but not affected by prazosin, propranolol, apamin or iberiotoxin. At higher concentrations (10 nM - 10 microM) UK 14,304 also induced a depolarizing effect which was potentiated by yohimbine and inhibited by prazosin. These results indicate that UK 14,304 acts on alpha(2)-adrenoceptors at lower concentrations and on both alpha(1)- and alpha(2)-adrenoceptors above 10 nM. In rings, with or without endothelium, noradrenaline had a depolarizing effect which was inhibited by prazosin. Adrenaline did not affect the membrane potential but in the presence of prazosin caused hyperpolarization, which was inhibited by yohimbine and glibenclamide. These results indicate that noradrenaline is more selective for alpha(1)-, whereas adrenaline has similar affinities for alpha(1)- and alpha(2)-adrenoceptors. In aortae with endothelium, L-NNA caused a small depolarization but did not affect the hyperpolarization induced by UK 14,304, indicating that NO is not involved in that response. Glibenclamide induced a small depolarization in aortae, with or without endothelium, indicating that ATP-sensitive K(+) channels may play a role in maintaining the smooth muscle's membrane potential. Our results indicate that, in rat aorta, alpha(2)-adrenoceptors are also present in the smooth muscle, and that these receptors act through small-conductance ATP-sensitive K(+) channels.

Effect of cholecalciferol treatment on the relaxant responses of spontaneously hypertensive rat arteries to acetylcholine.

We studied the effect of oral cholecalciferol treatment on the endothelium-dependent vascular relaxation and hyperpolarization induced by acetylcholine (ACh), which is impaired in spontaneously hypertensive rats (SHR). Adult female SHR and normotensive Wistar-Kyoto rat (WKY) controls received 125 microg of cholecalciferol per kilogram body weight per day for 6 weeks. The responses to ACh of the isolated mesenteric vascular bed and mesenteric artery rings were measured, as well as the smooth muscle cell membrane potential. After cholecalciferol treatment, the systolic blood pressure and basal perfusion pressure of the mesenteric vascular bed of the SHR fell to control levels. The relaxant and hyperpolarizing effects of ACh, which are reduced in SHR, were also brought to control levels after cholecalciferol treatment. These effects of ACh were inhibited by N(omega)-nitro-L-arginine in SHR and by apamin in WKY. After cholecalciferol treatment, SHR hyperpolarizing responses showed the same inhibition pattern as those of WKY. This indicates that, after cholecalciferol treatment, SHR vascular mesenteric preparation responses to ACh are mediated by endothelium-derived hyperpolarizing factor, which induces activation of Ca(2+)-dependent K(+) channels, as in WKY. In untreated SHR, the ACh-mediated response is entirely due to ACh acting via the release of nitric oxide.

Recovery of impaired K+ channels in mesenteric arteries from spontaneously hypertensive rats by prolonged treatment with cholecalciferol.

1. The mechanism responsible for blood pressure reduction in spontaneously hypertensive rats (SHR) after prolonged cholecalciferol treatment was studied. Two-week treatment of SHR with 0.125 mg cholecalciferol kg-1 body weight per day orally caused significant reductions of systolic blood pressure and of the resting perfusion pressure of the mesenteric vascular bed at constant flow. 2. In addition, the treated animals presented a normalization of the maximum vasoconstriction response to noradrenaline and a reduction of the maximum effect of the adrenaline concentration-response curves. This latter effect probably was due to recovery of the impaired Ca(2+)-dependent K+ channels coupled to alpha 2-adrenoceptors since it was prevented by apamin. 3. The treatment with cholecalciferol also normalized the smooth muscle cell membrane potential of de-endothelialized mesenteric arteries of SHR and their hyperpolarizing responses to alpha 2-adrenergic agonists, which were depressed in untreated SHR. 4. In mesenteric rings with endothelium, alpha 2-adrenergic agonists caused similar hyperpolarizing responses in the SHR and in normotensive Wistar (NWR) and Wistar Kyoto (WKY). In non cholecalciferol-treated SHR the hyperpolarizing mediator involved in this effect was NO, while in NWR it was the endothelium-derived hyperpolarizing factor (EDHF). After cholecalciferol treatment, the hyperpolarization induced by alpha 2-adrenergic agonists in SHR smooth muscle cells was mediated by EDHF, as in NWR. 5. Our results indicate that the hypotensive effect of cholecalciferol in the SHR is probably due to the normalization of vascular reactivity, by restoring the functioning of apamin- and ATP-sensitive K+ channels located in the vascular smooth muscle cell membrane, which are impaired in the SHR.

Impaired function of alpha-2 adrenoceptors in smooth muscle of mesenteric arteries from spontaneously hypertensive rats.

The alpha2-adrenoceptor function in mesenteric arteries of spontaneously hypertensive rats (SHR) was investigated by comparing membrane potential changes in response to adrenergic agonists in preparations from female SHR, Wistar-Kyoto (WKY) and normotensive Wistar rats (NWR). Resting membrane potential was found to be less negative in mesenteric arteries from SHR than in those from NWR and WKY. Apamin induced a decrease in the membrane potential of mesenteric artery rings without endothelium from NWR and WKY, but had no effects in those from SHR. Both UK 14,304 and adrenaline, in the presence of prazosin, induced a hyperpolarization that was significantly lower in de-endothelialized mesenteric rings from SHR than in those from NWR and WKY. In mesenteric rings with endothelium, however, similar hyperpolarization was observed in the three strains. In NWR mesenteric rings with endothelium the hyperpolarization induced by activation of alpha2-adrenoceptors was abolished by apamin, whereas in intact SHR mesenteric rings this hyperpolarization was slightly reduced by apamin and more efficiently reduced by Nomega-nitro-L-arginine. It is concluded that the activity of potassium channels coupled to alpha2-adrenoceptors is altered in the smooth muscle cells of SHR mesenteric arteries, contributing to their less negative membrane potential. On the other hand, the endothelial alpha2-receptors are functioning in mesenteric vessels from SHR and their stimulation induces a hyperpolarization mainly through the release of nitric oxide.

Dual effect of clonidine on mesenteric artery adrenoceptors: agonistic (alpha-2) and antagonistic (alpha-1).

The effect of clonidine on the mesenteric vascular bed and the isolated mesenteric artery was examined in preparations in which tonus was induced by norepinephrine or endothelin. In preparations precontracted by norepinephrine, clonidine caused a relaxation which was not inhibited by the alpha-2 antagonists yohimbine and idazoxan or by the K+ channel blockers apamine, tetraethylammonium and glibenclamide. In preparations precontracted with endothelin, clonidine increased the depolarization and induced a contraction. Both these effects were inhibited by prazosin. In isolated mesenteric arteries, norepinephrine cause a significant depolarization that was inhibited by clonidine or prazosin. On the other hand, clonidine caused a hyperpolarization which was inhibited by idazoxan or yohimbine, but not by prazosin. This hyperpolarization was also abolished by apamine, tetraethylammonium and glibenclamide. It is concluded that clonidine acts on alpha-1 adrenoceptors as a partial agonist, causing relaxation of the mesenteric artery precontracted with norepinephrine or contraction of preparations precontracted with endothelin. Moreover, clonidine can open K+ channels and hyperpolarize the plasma membrane of mesenteric artery by acting on alpha-2 adrenoceptors.

Mechanism of smooth muscle contraction and relaxation mediated by kinin receptor.

The increase in sensitivity of guinea pig preparations to bradykinin (BK) due to stretching occurring with time after mounting was studied by determining the time course of changes in the cell membrane potential, measured with intracellular microelectrodes. A sustained hyperpolarizing effect of BK, which was observed in recently mounted preparations, became transient after 120 min, when it was followed by depolarization, which was much more evident after 4 h of stretching. As a consequence, a parallel increase in the contractile response to BK was also observed. The hyperpolarizing effect was due to the opening of Ca(2+)-dependent K+ channels sensitive to apamin, since BK dose-response curves done within 1 h of mounting were shifted to the left, becoming similar to dose-response curves obtained 4 h after mounting of the guinea pig ileum preparation. These results were specific for BK, since the potentiating effect of apamin was not observed for acetylcholine. Our results show that the activation of B2 receptors by BK in the isolated guinea pig ileum induce a dual effect--hyperpolarization and depolarization--and that the increase in the contractile response consequent to stretching is probably due to the inactivation of apamin-sensitive Ca(2+)-dependent K+ channels.

Mechanism of smooth muscle contraction and relaxation mediated by kinin receptor

The increase in sensitivity of guinea pig preparations to bradykinin (BK) due to stretching occurring with time after mounting was studied by determining the time course of changes in the cell membrane potential, measured with intracellular microelectrodes. A sustained hyperpolarizing effect of BK, which was observed in recently mounted preparations, became transient after 120 min, when it was followed by depolarization, which was much more evident after 4 h of stretching. As a consequence, a parallel increase in the contractile response to BK was also observed. The hyperpolarizing effect was due to the opening of Ca²+-dependent K+ channels sensitive to apamin, since BK dose-response curves done within 1 h of mouting were shifted to the left, becoming similar to dose-response curves obtained 4 h after mounting of the guinea pig ileum preparation. These results were specific for BK, since the potentiating effect of apaming was not observed for acetylcholine. Our results show that the activation of B2 receptors by BK in the isolated guinea pig ileum induce a dual effect - hyperpolarization and depolarization - and that the increase in the contractile response consequent to stretching is probably due to the inactivation of apaminsensitive Ca²+-dependent K+ channels

BK1 and BK2 bradykinin receptors in the rat duodenum smooth muscle.

1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.

Effect of treatment with vitamin D3 on the responses of the duodenum of spontaneously hypertensive rats to bradykinin and to potassium.

1. The diet of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) and Wistar (NWR) rats was supplemented with either 2% calcium lactate in the drinking water or 12.5 micrograms vitamin D3 100 g-1 body weight daily by gavage, for 14 days. 2. The blood pressure of the SHR treated with either calcium or vitamin D decreased to the same levels as that of WKY and NWR. 3. The response to bradykinin of the SHR isolated duodenum, which is predominantly contractile, upon treatment with vitamin D (but not with calcium), became predominantly relaxant, approaching the normal behavior of the WKY and NWR duodenum. 4. The relaxant responses of the SHR and WKY duodenum to potassium were smaller than those of NWR, but treatment with vitamin D increased the response in all three rat strains. 5. It is concluded that, besides sharing the hypotensive effect of calcium, vitamin D treatment of SHR has an effect on the duodenum smooth muscle which might be due to calmodulin-dependent activation of calcium-dependent potassium channels.

Mechanism of the relaxant response of the rat duodenum to bradykinin.

Bradykinin (BK) did not increase cyclic AMP production in cultured rat duodenum smooth muscle cells. Its relaxant effect on the tissue was inhibited by apamin and potentiated by phorbol dibutyrate (PDBU). PDBU also caused a relaxation which was inhibited by apamin. BK's relaxant effect, and its potentiation by PDBU, are due to activation of Ca(2+)-dependent K+ channels.

Angiotensin II desensitization and Ca2+ and Na+ fluxes in vascular smooth muscle cells.

The role of ion fluxes in angiotensin II (AII) desensitization (tachyphylaxis) was investigated by studying Na+ and Ca2+ translocation in cultured vascular smooth muscle cells from the rat aorta. The effects of AII were compared to those of [1-sarcosine]-AII (Sar1-AII), an analogue which also induces tachyphylaxis, and [2-lysine]-AII (Lys2-AII), an analogue that does not show this property. Maximally effective concentrations of the three peptides induced a rapid and transient increase in 45Ca2+ efflux, a rapid and sustained decrease in total cell Ca2+ and an increased Na+ permeability. Repeated treatments, at short intervals, with either of the three peptides abolished the effect on Ca2+ efflux, and this desensitization was slowly reversible. A 30-min rest period was sufficient for full recovery of the response of cells that were desensitized by Lys2-AII, whereas the recovery from AII or Sar1-AII-desensitization was still not complete after 60 min. Our results suggest that the difference in the behaviour of the "tachyphylactic" AII and Sar1-AII and the "non-tachyphylactic" Lys2-AII lays not in the production of different signals upon binding to the receptor, but in a difference in the hormone-receptor interaction itself.