Teresa Feres

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

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. 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 Ca2+‐dependent K+ channels coupled to α2‐adrenoceptors since it was prevented by apamin. The treatment with cholecalciferol also normalized the smooth muscle cell membrane potential of de‐endothelialized mesenteric arteries of SHR and their hyperpolarizing responses to α2‐adrenergic agonists, which were depressed in untreated SHR. In mesenteric rings with endothelium, α2‐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 α2‐adrenergic agonists in SHR smooth muscle cells was mediated by EDHF, as in NWR. 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.

Different mechanism of LPS‐induced vasodilation in resistance and conductance arteries from SHR and normotensive rats

The direct and endothelium‐dependent effects of lipopolysaccharide (LPS) were investigated on resistance and conductance arteries from normotensive Wistar (NWR) and spontaneously hypertensive (SHR) rats. In both NWR and SHR, LPS induced dose‐dependent relaxations of the mesenteric vascular bed, which were inhibited by L‐NNA in SHR but not in NWR. Iberiotoxin (IBTX) inhibited the responses to LPS in both groups, indicating the participation of high conductance Ca2+‐dependent K+ channels. In mesenteric artery rings, the resting membrane potentials and the hyperpolarizing responses of NWR to LPS did not differ in endothelized and denuded preparations but L‐NNA inhibited the responses only in endothelized rings. These responses were reduced by bosentan, suggesting that endothelin release may mask a possible hyperpolarizing response to LPS. The hyperpolarizing responses to LPS were blocked by IBTX in both endothelized and de‐endothelized NWR rings. In the SHR only intact rings showed hyperpolarization to LPS, which was inhibited by IBTX and byL‐NNA. In SHR aortic endothelized or denuded rings, LPS induced hyperpolarizing responses which, in endothelized rings, were partially blocked by L‐NNA, by IBTX or by glibenclamide, but totally abolished by IBTX plus glibenclamide. No response to LPS was observed in NWR aortic rings. Our results indicate that LPS activates large conductance Ca2+‐sensitive K+ channels located in the smooth muscle cell membrane both directly and indirectly, through NO release from the endothelium in NWR, whereas NO is the major mediator of the LPS responses in SHR resistance vessels.

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

1 The α2‐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). 2 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. 3 In NWR mesenteric rings with endothelium the hyperpolarization induced by activation of α2‐adrenoceptors was abolished by apamin, whereas in intact SHR mesenteric rings this hyperpolarization was slightly reduced by apamin and more efficiently reduced by Nω‐nitro‐l‐arginine. 4 It is concluded that the activity of potassium channels coupled to α2‐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 α2‐receptors are functioning in mesenteric vessels from SHR and their stimulation induces a hyperpolarization mainly through the release of nitric oxide.

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 μg 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 behaviour 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.

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 Lys2AII, whereas the recovery from AII or Sar1AII-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.

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

The role of α2‐adrenoceptors in the response of aorta smooth muscle rings to the α2‐adrenoceptors agonists UK 14,304 and clonidine was studied. Stimulation by 1–10 nM UK 14,304 caused dose‐dependent relaxant responses in BaCl2‐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 μM) 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 α2‐adrenoceptors at lower concentrations and on both α1‐ and α2‐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 α1‐, whereas adrenaline has similar affinities for α1‐ and α2‐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 muscles membrane potential. Our results indicate that, in rat aorta, α2‐adrenoceptors are also present in the smooth muscle, and that these receptors act through small‐conductance ATP‐sensitive K+ channels.

Role of membrane potential and expression of endothelial factors in restenosis after angioplasty in SHR.

Abstract—We examined the roles played by impaired K+ channels, diminished nitric oxide (NO) production, endothelin release, and smooth muscle membrane potential in the increased restenosis observed in spontaneously hypertensive rat (SHR) carotid arteries after angioplasty. The SHR carotid was found to be less polarized than that of normotensive Wistar rats (NWR), and it was further depolarized by the &agr;2 agonist UK 14,304. This response was blocked by iberiotoxin, indicating that calcium-dependent K+ channels operate normally in the SHR carotid. Acetylcholine caused a hyperpolarization that was significantly smaller in SHR than in NWR carotids, indicating a deficient release of NO in the SHR. After angioplasty, SHR and NWR vessels were depolarized, returning to baseline after 10 days. In the SHR but not in the NWR the contralateral carotid was also depolarized, and this was prevented by the endothelin A/B receptor antagonist bosentan. After angioplasty, endothelin-1 plasma levels increased in both SHR and NWR, but the increase was significantly more prolonged in SHR. We found that the more pronounced restenosis observed in the SHR carotid after angioplasty is not due to impairment of calcium-dependent K+ channels but is related to the relatively depolarized vascular smooth muscles, involving endothelin release caused by reduced NO levels in that strain.

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 preparations 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.

Cholecalciferol treatment restores the relaxant responses of spontaneously hypertensive rat arteries to bradykinin

The vasodilation and hyperpolarization induced by bradykinin (BK) in the mesenteric vascular bed and mesenteric arteries from spontaneously hypertensive rats (SHR) and from normotensive Wistar rats (NWR), as well as Wistar Kyoto rats (WKY), was investigated before and after prolonged oral treatment with cholecalciferol (125 mg kg(-1) body weight per day) for 3 weeks. The cholecalciferol treatment caused a decrease in the SHR blood pressure, as well as a normalization in the resting potential of the smooth muscle cell membrane of mesenteric arteries and restored their hyperpolarizing response to BK. The concentration-response curves for the vasodilator effect of BK on the mesenteric vascular bed were significantly decreased in SHR and in WKY when compared with NWR. Cholecalciferol treatment improved the maximum responses of the SHR preparation, bringing them to levels similar to those of the NWR preparations, which themselves were unaffected by the treatment. In the presence of apamin, a Ca(2+)-dependent K(+) channel inhibitor, the maximum responses to BK in preparations from NWR or cholecalciferol-treated SHR decreased to values similar to those observed in untreated SHR. Our results indicate that the low responsivity of the SHR resistance vessels to the relaxant effect of BK is due to impaired Ca(2+)-dependent K(+) channels and that reversion of this impairment contributes to the blood pressure reduction caused by the cholecalciferol treatment. However, the mechanism of the low responsivity in WKY remains to be investigated.