Therezinha B. Paiva

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.

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum

1 Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. 2 The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 μM), but pre‐incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. 3 The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. 4 Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. 5 Des‐Arg9‐bradykinin, an analogue specific for B1‐receptors, was much less active than bradykinin, and elicited only a contractile response. 6 Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. 7 Our results support the hypothesis that the relaxant and contractile components of the rat duodenums response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.

Role of the two n-terminal residues of angiotensin II in the production of tachyphylaxis

The structural requirements for the production of angiotensin tachyphylaxis in the guinea-pig ileum were studied by analyzing the tachyphylactic properties of the following synthetic analogues of angiotensin II (AII): [1-sarcosine]AII, [1-betaine]AII; [1-guanidinoacetic]AII; betainyl-AII; [2-lysine]AII; [2-ornithine]AII. In the non-atropinized ileum, no tachyphylaxis was observed with any of the following analogues: [2-lysine]AII, [2-ornithine]AII, [2-ornithine]AII, [1-betaine]AII and betainyl-AII. [1-Guanidinoacetic]AII induced tachyphylaxis, but to a smaller degree than AII, while [1-sarcosine]AII was significantly more tachyphylactic than AII. Similar results were obtained in the atropinized ileum, except that moderate tachyphylaxis was also observed with betainyl-AII and [1-betaine]AII. The analogues with lysine or ornithine residues in position 2 did not induce tachyphylaxis under any of the conditions studied. It is concluded that, besides the protonated N-terminal amino group, the guanidino group of the Arg2 side-chain is essential for the manifestation of angiotensin tachyphylaxis in the guinea-pig ileum.

EFFECT OF INDOMETHACIN AND PROSTAGLANDIN ON THE SMOOTH MUSCLE CONTRACTING ACTIVITY OF ANGIOTENSIN AND OTHER AGONISTS

1 Indomethacin had an equal inhibitory effect on the response of the guinea‐pig isolated ileum to angiotensin II (angiotensin), bradykinin, histamine and acetylcholine. This effect did not seem to result from inhibition of prostaglandin synthesis, as it did not depend on the time of treatment with indomethacin. 2 Prostaglandin E2 (prostaglandin) potentiated the responses of the guinea‐pig ileum to angiotensin, bradykinin, histamine and acetylcholine without significant differences in the effects observed. 3 In the rabbit isolated mesenteric and coeliac arteries, indomethacin had an equal potentiating effect on the responses to angiotensin and to adrenaline. In these organs pre‐incubation with indomethacin was necessary for the effect to be observed, and this effect lasted for 2 h or more after that drug was removed from the medium. 4 No cross‐tachyphylaxis between angiotensin and adrenaline was observed in the rabbit mesenteric and coeliac arteries. 5 It is concluded that the effects of indomethacin and prostaglandin on the response of the guinea‐pig ileum to the four agonists result from an action on the smooth muscle contractile mechanism per se rather than from an inhibitory action on the release of endogenous prostaglandin produced by the four agonists. 6 The results with the rabbit isolated arteries indicate that tachyphylaxis to angiotensin in these organs is not caused by prostaglandin release.

The balloon catheter induces an increase in contralateral carotid artery reactivity to angiotensin II and phenylephrine

The effects of balloon injury on the reactivity of ipsilateral and contralateral carotid arteries were compared to those observed in arteries from intact animals (control arteries). Carotid arteries were obtained from Wistar rats 2, 4, 7, 15, 30 or 45 days after injury and mounted in an isolated organ bath. Reactivity to angiotensin II (Ang II), phenylephrine (Phe) and bradykinin (BK) was studied. Curves were constructed in the absence or presence of endothelium or after incubation with 10 μM indomethacin, 500 μM valeryl salicylate or 0.1 μM celecoxib. Phe, Ang II and BK maximum effects (Emax) were decreased in ipsilateral arteries when compared to control arteries. No differences were observed among pD2 or Hill coefficient. Emax to Phe (4 and 7 days) and to Ang II (15 and 30 days) increased in the contralateral artery. In addition, Phe or Ang II reactivity was not significantly different in aorta rings from control or carotid‐injured animals. The increased responsiveness of contralateral artery was not due to changes in carotid blood flow or resting membrane potential. The endothelium‐dependent inhibitory component is not present in the contraction of contralateral arteries and it is not related to superoxide anion production. Indomethacin decreased contralateral artery responsiveness to Phe and Ang II. Valeryl salicylate reduced the Ang II response in contralateral and control arteries. Celecoxib decreased the Phe Emax of contralateral artery. In conclusion, decreased endothelium‐derived factors and increased prostanoids appear to be responsible for the increased reactivity of contralateral arteries after injury.

The effect of pH on tachyphylaxis to angiotensin peptides in the isolated guinea pig ileum and rat uterus

Abstract Angiotensin tachyphylaxis in the isolated guinea pig ileum and rat uterus was studied with the aid of the following synthetic peptides: [Ile5]agiotensin II (A II), [Asn1] A II, [Gly1] A II, [Arg1] A II, des-Asp1-A II, [Gly1,Gly2] A II, [Suc1] A II and acetyl-A II. The pK values for the amino and imidazole groups of these peptides were determined by electrometric titration. The effect of pH on tachyphylaxis to the angiotensin analogs and homologs was studied in the two smooth muscle preparations. The intensity of tachyphylaxis produced by the peptides correlated well with the degree of protonation of the amino group, but not with that of the imidazole group or with the net charge of the peptide. [Gly1,Gly2] A II and the two peptides in which the amino group was absent ([Suc1] A II) or blocked (acetyl-A II) did not produce any tachyphylaxis in the guinea pig ileum or the rat uterus. It is concluded that the protonated amino group plays an important role in the phenomenon of tachyphylaxis, in both organs, and that the guanido group is also important.

Evidence for a regulatory site in the angiotensin II receptor of smooth muscle.

The homologous desensitization induced by angiotensin II analogues in the guinea-pig isolated ileum was studied. Desensitization assessed by the loss of response on repeated treatment showed [Sar1]angiotensin II to be a strong desensitizer whereas no desensitization to [Lys2]angiotensin II was detected. However, prolonged treatment with either analogue desensitized the tissue, indicating that [Lys2]angiotensin II-induced desensitization was reversed faster. A correlation was found between the degree of desensitization caused by repeated treatment and the time for half-relaxation after washout of the first treatment, but the relaxation after washout became faster in the desensitized state. In experiments designed to study competition between the agonistic and desensitizing properties of angiotensin II analogues, high concentrations of [Lys2]angiotensin II blocked the agonistic but not the desensitizing effect of lower concentrations of [Sar1]angiotensin II. It is concluded that desensitization is due to the interaction of angiotensin II with a regulatory site on the receptor.

Role of Ca+‐dependent K‐channels in the membrane potential and contractility of aorta from spontaneously hypertensive rats

1 Contractile responses to KCl and membrane potentials were determined in aortic rings from spontaneously hypertensive rats (SHR), normotensive Wistar rats (NWR) and Wistar Kyoto rats (WKY) both in the absence and in the presence of the Ca2+‐dependent K‐channel blockers, apamin and tetraethylammonium (TEA). 2 Compared to NWR, aortic rings from WKY and SHR were less reactive and their Ca2+ uptake after stimulation with K+ was decreased. 3 Smooth muscle cell membrane potentials were higher in aortae from SHR and WKY than in NWR aortae, whereas SHR had higher K+ and lower Na+ intracellular activities than WKY and NWR, suggesting overactivity of the Na+/K+ pump in the hypertensive animals. 4 Treatment with apamin caused depolarization of WKY and SHR aortae, and increased their contractile responses to the same level as those of the NWR. Treatment with TEA also caused depolarization of aortae from WKY and SHR, but in the SHR the depolarization induced by TEA was smaller than that produced by apamin and the contractile responses to KCl did not reach the level of those of aortae from NWR. 5 It is concluded that overactivity of Ca2+‐dependent K‐channels in aortae of WKY and SHR contributes to their higher membrane potentials and lower responsiveness to vasoconstrictor stimuli. In SHR, an overactive Na+/K+ pump is also present, and the contribution of apamin‐sensitive Ca2+‐dependent K‐channels to the membrane potential and reactivity appears to be more relevant than that of TEA‐sensitive channels.

Endothelium-dependent inhibition of the use of extracellular calcium for the arterial response to vasoconstrictor agents

The responses of rabbit mesenteric or coeliac artery rings to angiotensin II or adrenaline (but not to K+) were enhanced by endothelium destruction (by rubbing). Potentiation by indomethacin of the response to the agonists was observed in rubbed rings but not in intact ones. Both angiotensin II and adrenaline (in the presence of propranolol and prazosin) induced endothelium-dependent relaxation of the arteries. Rubbed rings, but not intact ones, contracted when Ca2+ was added to a previously Ca2+-free medium containing angiotensin II or adrenaline. The vasoconstrictor response appears to be modulated by the regulation of receptor-operated Ca2+ channels through EDRF released by the endothelium and by some cyclo-oxygenase product at the level of the smooth muscle cell membrane.