Occurrence of CGRP receptors on dedifferentiated smooth muscle cells from media of rat thoracic aorta.

Autoradiographic studies with [125I]-CGRP did not demonstrate receptors on sections from two different regions of 5-week-old rat aorta (of both sexes). By contrast, cultured smooth muscle cells isolated from similar aortic media and passaged 5 to 7 times exhibited specific binding.

Schistosoma mansoni: the chemical nature of the secretions produced by the Mehlis' gland and ootype as revealed by cytochemical studies.

The 'secretory pathway' of the Mehlis' gland secretion as well as the maturation of the secretory granules are described. The secretions produced by the Mehlis' gland and the distal ootype in Schistosoma mansoni were identified as neutral glycoproteins. They were both periodate reactive, stained with phosphotungstic acid at low pH, and sensitive to the proteolytic action of papain. The secretions did not strain with both the low-iron diamine and the high-iron diamine methods for the presence of acidic glycoconjugates, and they did not contain a detectable level of sulphydryl groups. The possible role of the secretion from the Mehlis' gland in egg-shell formation is discussed.

Microvascular effects of bradykinin in the isolated perfused guinea-pig hindbrain: method and characterization of a possible third type of bradykinin receptor.

The method used by McGregor for the isolated mesenteric vascular bed was adapted to study the mechanisms of regulation of cerebral blood flow in the isolated perfused guinea-pig brain. This method allows the recording of the perfusion pressure in the hindbrain perfused at constant physiological flow, and has been successfully applied to the study of the effect of bradykinin. Bradykinin elicited a vasoconstriction that was independent of the presence of the endothelium. The pD2 ranged between 7.50 and 7.60, respectively, with and without the endothelium. Des-Arg9-BK was unable to elicit a vasoconstriction. [Tyr(Me)8]BK had a partial agonist activity. Both [Leu9]des-Arg10-KD and [DArg0,Hyp3,DPhe7]BK had similar antagonist activities. From these pharmacological characteristics, the receptor involved in this response seems to be different from a B1 or a B2 receptor.

Effects of substance P, calcitonin gene-related peptide and capsaicin on tension and membrane potential of pig coronary artery in vitro.

The effects of substance P (SP), calcitonin gene-related peptide (CGRP) and capsaicin were studied on isometric tension and membrane potential of pig coronary arterial strips in vitro. CGRP induced an endothelium-independent relaxation without change in the smooth muscle membrane potential whereas SP relaxed and hyperpolarized the strip via the endothelium. Applied together, the mechanical effects of SP plus CGRP were additive. CGRP did not affect the hyperpolarization due to SP. In order to examine a possible role of endogenous SP and CGRP, capsaicin was used. It provoked a contraction which was adventitia-dependent, and was inhibited by indomethacin. In presence of indomethacin, capsaicin caused a relaxation. It was accompanied by a hyperpolarization of smooth muscle membrane potential only when the strip had an intact endothelium. When the strip was de-endothelialized capsaicin relaxation subsisted. This indicates that capsaicin produced a relaxation of indomethacin-treated strip by releasing a hyperpolarizing endothelial factor and probably endogenous CGRP.

Effects of neurokinins on the isolated pig coronary artery.

The actions of substance P (SP), neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), kassinin (KAS) and eledoisin (ELE) were investigated on transversally cut strips of pig coronary artery. All tachykinins produced vasodilatation of coronary arteries precontracted with ACh; 10(-5) M. The order of potency was: SP = PHY ELE greater than KAS greater than NKA greater than NKB, with the ED50 values being 0.38, 0.38, 1.2, 2.6, 8.3 and 34.0 nM, respectively. Continued superfusion of SP (7.4 X 10(-9) M) desensitized the coronary arteries which were refractory to the vasodilator action of NKA, NKB, PHY and KAS. The arteries nevertheless dilated upon the addition of noradrenaline (NA) and bradykinin (BK). Endothelium-removed preparations did not respond to any of the tachykinins. However, tissues devoid of endothelium relaxed in response to both NA and vasoactive intestinal polypeptide (VIP). Three octapeptide antagonists, [D-Pro4,Ala6,D-Trp7,9,Nle11]SP-(4-11) (compound I), [D-Pro4,Ser6,D-Trp7,9,Nle11]SP-(4-11) (compound II) and [D-Pro4,D-Trp7,9,10,Phe11]SP-(4-11) (compound III) were examined as potential antagonists of tachykinin-induced vasodilatation. Compounds I and II blocked the actions of SP and NKA but not that of PHY. Compound III effectively blocked the actions of SP and PHY. We conclude that the pig coronary artery possesses a 'NK-P/SP-P' type receptor, and that this receptor is probably localized on the endothelium.

Interaction of bradykinin and des-Arg9-bradykinin with isolated pig coronary arteries: mechanical and electrophysiological events.

This paper describes the effect of bradykinin (BK) and des-Arg9-BK on the isometric tension and smooth muscle membrane potential of transverse strips of pig coronary artery. BK causes a relaxation of contracted muscle. This effect is particularly evident in muscle which has previously been contracted by acetylcholine. The relaxation is accompanied by a transient hyperpolarization of the vascular smooth muscle. Des-Arg9-BK, in contrast, causes a contraction of the muscle which is not accompanied by a significant change of transmembrane potential. The relaxing action of BK depends on the presence of the endothelium. In a "cascade" experiment, evidence is presented that a relaxing factor is released by the endothelium in response to BK. Thus the perfusate from a BK-stimulated intact artery can cause the relaxation of a pre-contracted de-endothelialized artery. We conclude that the endothelium has B2-receptors which cause the release of a humoral factor which hyperpolarizes and relaxes the muscle. The contracting action of des-Arg9-BK does not depend on the endothelium and appears to be mediated through B1-receptors directly on smooth muscle by pharmacomechanical coupling.

The rat isolated portal vein: a preparation sensitive to neurokinins, particularly neurokinin B.

The rat isolated portal vein is a pharmacological preparation more sensitive to neurokinin B than to any other neurokinin or tachykinin. The preparation is more sensitive to C-terminal partial sequences of substance P (SP) particularly SP-(6-11) than to the whole undecapeptide. The order of potency of neurokinins is as follows: neurokinin B greater than neurokinin A greater than substance P. The preparation shows high sensitivity also to kassinin and eledoisin. Comparative tests performed with strips of the rat portal vein suspended in a microbath under continuous perfusion (system 1) or in ordinary baths for isolated smooth muscles (system 2) have given similar results and have shown that the myotropic effect of neurokinin B is not modified by a variety of antagonists of endogenous agents as well as by inhibitors of the arachidonic acid cascade. The present results suggest that neurokinin B contracts the rat portal vein by activating specific receptors, presumably located on the smooth muscle membrane, different from those of biologically active amines and peptides which are active stimulants of the vein. Neurokinin B is ten times more active than neurokinin A and at least 100 times more than substance P. Such an order of potency of agonists suggests the existence of a new neurokinin receptor type, particularly sensitive to neurokinin B.

Effect of mechanical stimulation, substance P and vasoactive intestinal polypeptide on the electrical and mechanical activities of circular smooth muscles from pig coronary arteries contracted with acetylcholine: role of endothelium.

Mechanical stimulation, substance P and vasoactive intestinal polypeptide (VIP) were found to relax the transversal strip of anterior descending branches of pig coronary arteries precontracted by acetylcholine. The effects of mechanical stimulation and substance P required the presence of intact endothelium, while VIP did not. The effect of VIP did not appear to be mediated by catecholamines. Simultaneous measurements of intracellular membrane potential and tension developed by coronary smooth muscle precontracted with Ach showed that the smooth muscle relaxation by substance P is accompanied by membrane hyperpolarization. In contrast VIP relaxed the same tissue without affecting the membrane potential. In a cascade experiment, the fluid perfused intraluminally in intact segments of coronary arteries was dropped over a de-endothelialized strip which relaxed in response to substance P and mechanical stimulation. This indicates that substance P and mechanical stimulation act by releasing from the endothelium a humoral factor that produces arterial smooth muscle relaxation.

Tachykinin antagonists: substitutions in positions 5 and 6 with amino acids from the primary sequence of substance P homologues.

A series of tachykinin antagonists has been synthetized by substituting the amino acids of eight naturally occurring tachykinins into the general antagonist [pro4, trp7,9, Phe 11]tachykinin-(4-11). Five decapeptide antagonists were also synthetized. These antagonists were tested on four smooth muscle preparations, the rabbit mesenteric vein, the guinea-pig ileum, the guinea-pig trachea and the rat urinary bladder. On all tissues, except the rat urinary bladder, antagonists that had amino acids other than Gln5 Gln6 found in the substance P molecule were inactive as antagonists, and some had marked intrinsic activity on the guinea-pig ileum and the guinea-pig trachea. The inhibitory activity of these antagonists on the rat urinary bladder, however, was quite marked. The activities of these antagonists on the rat urinary bladder can be summarized as follows: (a) In general decapeptide antagonists were of low affinity. (b) Octapeptide antagonists showed variable affinities against the various tachykinins and some were selective. The only two antagonists which were fairly active against all tachykinins were [pro4, trp7,9, Phe11]SP-(4-11) and [pro4, trp7,9, Phe11] UPE-(4-11). (c) Physalaemin was frequently antagonized in a non-surmountable manner. (d) Eledoisin and kassinin were each inhibited by only one antagonist, and the antagonist was different for each tachykinin. Some tachykinin receptors on smooth muscle have a binding site which is highly selective for Gln5 Gln6, especially if the affinity of antagonists is considered. Another tissue, the rat urinary bladder, does not exhibit this selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacodynamic studies with flunarizine, a calcium influx blocker.

Flunarizine, a difluoro derivative of cinnarizine, has been used to study its antivasoconstrictor effects on isolated perfused rat mesentery. The vasoconstrictor responses induced by sequential intra-arterial injections of phenylephrine (PhE) and norepinephrine (NE), were measured as an increase in the perfusion pressure (mm Hg). Flunarizine was administered either by i.v. route in a dose of 3 mg/kg, 15-20 min before the isolation of the mesentery or it was added to the perfusing medium, in concentrations ranging from 100 pg to 1 microgram/ml (PhE experiments) and from 1 ng to 1 microgram/ml (NE experiments). Intravenous administration of flunarizine significantly decreased the vascular reactivity of rat mesentery to PhE and NE at all dose levels. Continuous perfusion of flunarizine attenuated the vasoconstrictor responses induced by PhE and NE. Flunarizine was further studied for its hemodynamic effects in anaesthetized rats using the radioactive microspheres technique. Flunarizine (5 mg/kg i.p.) was administered 60 min prior to the determination of hemodynamic parameters. It was found not to affect general hemodynamics (mean blood pressure, heart rate, cardiac output and hematocrit). However, it produced a significant decrease of renal and splenic blood flows. The changes in blood flows were associated with similar changes in the percentage distribution of cardiac output to these organs, thus indicating that local regulatory mechanisms were influenced by flunarizine. It appears that flunarizine has preferential effects on the different vasculatures in vivo, whereas it produces nonspecific inhibition of exogenous PhE and NE in the rat mesentery in vitro.

Effects of leukotriene C4 and prostaglandin E2 on the rat mesentery in vitro and in vivo.

Leukotriene C4 (LTC4) and Prostaglandin E2 (PGE2) have been studied for their effects on the rat mesentery in vitro and in vivo. Their effects on the norepinephrine (NE) induced vasoconstrictions in the above vascular bed have also been investigated. LTC4 (10(-10)M) and PGE2 (2.8 X 10(-7)M) produced no direct effects on the perfusion pressure in the isolated perfused rat mesentery. However, LTC4 (10(-10)M) produced constriction of the arterioles in vivo. PGE2 (2.8 X 10(-7)M) did not produce any direct effect on the microcirculation. Indomethacin, when superfused for 10 min. in a concentration of 7 X 10(-5)M, produced significant arteriolar constriction; this effect was reversed when PGE2 was added to the superfusing media containing indomethacin. LTC4, perfused in a concentration of 10(-10)M, produced a time - dependent inhibitory effect on NE induced vasoconstrictions in vitro whereas PGE2 perfusions (2.8 X 10(-7)M) potentiated the NE responses. This potentiating effect of PGE2 was inhibited by indomethacin. In contrast, the topical application of PGE2 in vivo attenuated NE responses of the mesenteric arterioles.

Postjunctional localization of substance P receptors on the rat portal vein.

A dose-dependent contractile effect of substance P (SP) on the isolated, everted rat portal vein was competitively inhibited by two selective SP antagonists (pro2, phe7, trp9)-SP and (pro4, trp7,9)-SP 4-11. Phentolamine, atropine, methysergide, mepyramine, cimetidine, Sar1, Ile8-angiotensin II, Leu8, des-Arg9-bradykinin and indomethacin did not block the action of SP. However, some of these antagonists differentially reduced SP responses, but such inhibitory effects were shown to be nonspecific. The results suggest that the SP-induced contractions of the rat portal vein were directly mediated by specific receptors localized on the smooth muscle cells. In addition, the response to SP appeared to be independent of prostaglandin biosynthesis.

Pharmacological and hemodynamic studies on flunarizine.

Systemic administration of flunarizine (3 mg/kg i.v.) to rats decreased the vascular reactivity of their mesenteries to phenylephrine (PhE) and norepinephrine (NE). Intra-arterial perfusion of flunarizine (100 pg to 1 microgram/ml) produced dose dependent attenuation of the vasoconstrictor responses induced by PhE and Ne in the isolated perfused rat mesentery. Flunarizine (5 mg/kg i.p.) did not significantly affect general hemodynamics (i.e. mean blood pressure, heart rate, cardiac output and hematocrits) in anaesthetized rats. However, it produced significant decreases in the blood flows to the kidneys and the spleen associated with similar changes in the percentage distribution of the cardiac output to these organs. These studies indicate that in rats, flunarizine produced preferential effects in different vascular beds.

Substance P-containing nerve fibres in large peripheral blood vessels of the rat.

Substance P-immunoreactive nerve fibres were localized by the indirect immunohistochemical method in the adventitia and the adventitial-medial border of large peripheral arteries and veins of the rat. Arteries showed a richer substance P-containing innervation than veins. The superior mesenteric artery was densely innervated, whereas no substance P-containing fibres were found around the carotid artery. Substance P produced a vasoconstriction of the veins, but was basically without effect on arteries, although with the carotid artery a dose-dependent relaxation was observed. The absence of a correlation between the degree of innervation of the blood vessels and their responsiveness to exogenous substance P suggests that there nerves do not subserve a vasomotor function. The depletion of substance P immunoreactivity from nerves in arteries and veins by capsaicin suggest that substance P-containing vascular nerves are primarily sensory in nature.

Modulatory effects of substance P on norepinephrine induced vasoconstrictor responses in the rat mesentery.

1. The role of substance P (SP) in adrenergic transmission has been studied in the isolated perfused rat mesentery. 2. Intra-arterial perfusion of SP (4 x 10(-9) to 4 x 10(-7)M) produced a dose dependent potentiation of norepinephrine (NE) induced vasoconstrictor responses and a shift of the log dose--response curve to the left. 3. Saralasin, a specific antagonist of angiotensin II (ang. II) when perfused at a concentration of 3 x 10(-9)M did not change the NE responses as such. However, the potentiating effect of SP could not be demonstrated in the presence of saralasin. 4. Indomethacin, a prostaglandin (PG) synthetase inhibitor, when perfused at a concentration of 2.8 x 10(-6)M, markedly attenuated the vascular responses to NE, which could not be normalized by the addition of SP. 5. It appears that SP's potentiating effect on NE responses in the perfused rat mesentery simulates ang. II and not prostaglandins. A direct non-specific post synaptic sensitizing effect of SP on vascular smooth muscle cell, for its potentiating property, cannot be ruled out.

Effects of captopril (SQ 14225) on norepinephrine-induced vasoconstriction in the isolated perfused mesentery and hindquarters of the rat.

Captopril (SQ 14225) is an orally active agent that inhibits the conversion of ang. I to ang. II and also potentiates the biological actions of kinins. This substance has been used to study the role of ang. II/kinins on the norepinephrine-induced vasoconstriction in the isolated perfused mesentery and hindquarters of the rat. The vasoconstrictor responses were measured as an increase in the perfusion pressure (mm Hg), induced by sequential injections of norepinephrine. Captopril/placebo (physiological saline solution) was administered in a dose of 1 mg/kg by oral route, 15-20 min before isolating the vascular beds from these rats. Oral administration of captopril, decreased the vascular reactivity to norepinephrine in both the vascular beds, when compared to the responses obtained in the placebo treated rats. In another series of experiments (normal rats) bradykinin (BK), at concentrations of 0.4 and 0.8 micrograms/ml in the perfusate, attenuated the norepinephrine-induced vasoconstriction dose dependently, only in the mesenteric bed. It may be concluded that captopril attenuated the vascular reactivity to norepinephrine either by decreasing ang. II formation and/or increasing the action of kinins in the vicinity of these two beds. The latter mechanisms, however, appears not to play any direct role in the hindquarter bed. The direct effects of captopril on the decreased vascular reactivity of these beds cannot, however, be ruled out.

Effect of labetalol on adrenoceptors in the isolated perfused rat mesentery.

Isolated perfused rat mesentery has been used to define alpha- and beta-adrenoceptors. Norepinephrine was used as a reference substance to induce vasoconstrictor responses (increase in perfusion pressure). Log-dose response curves were obtained in the dose range between 0.3-10 mug. Labetalol, a known competitive inhibitor of both alpha- and beta-adrenoceptors, when administered as a single bolus injection (3 mug) initially induced predominant alpha-blockade lasting upto 75 min followed by potentiation of the vasoconstrictor responses to norepinephrine used at multiple dose levels. This potentiation was maximum between 150-195 min after injection of labetalol. This potentiating effect of labetalol observed in the later phase of the experiment might be related to its inhibitory mechanism of neuronal uptake.