Atrial natriuretic peptide (ANP) does not inhibit the basal vascular tone present in the in situ blood-perfused dog gracilis muscle

This study evaluated the effects of rat ANP(5-28) infusion into the blood-perfused dog gracilis muscle at concentrations ranging from 30 to 10,000 pg/ml. The vasculature of gracilis muscles from anesthetized beagle dogs was isolated and pump-perfused at constant flow with blood utilizing an extracorporeal circuit. Maximal vasodilatory capacity was determined by adenosine injection. ANP was infused into the arterial circuit to produce increasing arterial blood concentrations. Each infusion lasted 10 min. Systemic arterial pressure, central venous pressure, cardiac output and heart rate did not change during ANP infusion into the gracilis vasculature. ANP at arterial blood concentrations up to 10,000 pg/ml did not produce significant vasodilation although the vasculature showed pronounced vasodilation in response to adenosine. In vitro experiments showed that ANP had much less vasorelaxant activity in dog femoral artery and saphenous vein than in rabbit aorta. Therefore, rat ANP(5-28) at concentrations within and well above physiological and pharmacological ranges does not inhibit the basal vascular tone present in the innervated, blood-perfused dog gracilis muscle in situ.     

Electrophysiological analysis of the action of kavinton on the smooth muscles

Cavinton at a concentration of 10(-7)-10(-5) M was found to have a dose-dependent relaxing effect on bovine cerebral artery smooth muscles, without changing the resting potential and membrane resistance. Smooth muscles of the rabbit portal vein and guinea-pig taenia coli were insensitive to low cavinton concentrations. The results are consistent with the hypothesis that relaxing action of cavinton is due to the blocking of Ca2+ ions influx into the cells of cerebral artery through receptor-operated calcium channels. At higher concentrations (exceeding 10(-5) M) cavinton exerts nonspecific influence on the smooth muscles under study, inhibiting their excitability and decreasing membrane resistance resulting in the attenuation of tetanic contractions in the smooth muscles of the portal vein and taenia coli.     

In vitro receptor specificity of the 5HT1A selective phenylpiperazine, LY165163

LY165163, a ligand reported to be selective for the 5HT1A subtype of serotonin receptor, was examined for its ability to interact with 5HT2 receptors in the rat jugular vein and alpha-receptors in the rat aorta. In these smooth muscle preparations, no agonist activity of LY165163 occurred in concentrations up to 10(-5) M. However, LY165163 was an antagonist of serotonin-induced contractions in the jugular vein and of norepinephrine-induced contractions in the rat aorta. The dissociation constant calculated for LY165163 at 5HT2 receptors in the rat jugular vein was 10(-8) M and at alpha-receptors in the rat aorta was 2 X 10(-7) M. Thus, LY165163 is a relatively potent antagonist at vascular 5HT2 sites and possesses appreciable affinity at alpha-receptors. Based on these data, the multiple receptor interactions of LY165163 must be taken into consideration when utilizing this agent as a probe for the 5HT1A subtype of serotonin receptor.     

Myosin light chain phosphorylation and evidence for latchbridge formation in norepinephrine stimulated canine veins

The role of 20000 dalton myosin light chain phosphorylation in mediating venous smooth muscle contraction was studied in isolated preparations of canine jugular and femoral vein. One min 10(-5) M norepinephrine-induced contraction was accompanied by significant increases in phosphorylation (jugular - 21 to 46%; femoral - 19 to 54%) which were reversed within 10 min after agonist washout. During 40 min stimulation, phosphorylation and isometric force redevelopment rates declined to near basal levels while force was maintained. These findings implicate light chain phosphorylation as a prerequisite for initial tension development by crossbridge cycling in venous smooth muscle. However, long term tension can be maintained through a process similar to the latchbridge state in tracheal and arterial smooth muscle.     

Norepinephrine, monoamine oxidase, and acetylcholinesterase in the rat jugular vein compared with other blood vessels

The observation that the rat jugular vein relaxed in response to norepinephrine but not to field stimulation prompted us to evaluate the extent of innervation in this tissue. The norepinephrine concentration in the jugular vein was about 10% of that in the mesenteric artery and vein. The low levels of norepinephrine were not due to higher monoamine oxidase activity relative to the enzyme activity in other blood vessels. In the jugular vein, as in heart and brain, serotonin was preferred substrate for monoamine oxidase whereas in the femoral vein, mesenteric vein, and mesenteric artery, phenylethylamine oxidation was greater. Based on kinetic and inhibitory studies with LY51641, a selective type A inhibitor, monoamine oxidase activity was not found to be uniform throughout the cardiovascular system. In addition to low levels of norepinephrine, acetylcholinesterase activity in the jugular vein was only 5 and 13% of the activity in the portal vein and mesenteric artery, respectively. Thus, we provide strong evidence that our inability to generate a response to field stimulation in the rat jugular vein results from the lack of functional innervation in this tissue. This information adds to the usefulness of this preparation for comparative studies of agents acting on the smooth muscle without the added complication of neuronal uptake mechanisms.     

Quantitative and qualitative heterogeneity in smooth muscle myosin heavy chains

Previous studies have shown that smooth muscle myosin consists of two heavy chains (MHCs) of unequal molecular weight; however, it is not clear whether there are intermuscle, inter- and intraspecies differences in the MHCs. The purpose of these experiments was to quantitatively and qualitatively compare MHCs in different smooth muscles. Extracts of bovine aorta (BAo), dog saphenous vein (dSV) and femoral artery (dFA), and rat aorta (rAo), femoral artery (rFA), carotid artery (rCA), ileum (rGI) and uterus (rUt) were electrophoresed on 5% polyacrylamide-1% SDS gels. All tissues exhibited two MHCs with molecular weights of 207,000 (MHC1) and 204,000 (MHC2) daltons. In all cases the proportion of total MHC made up by MHC1 was greater than that by MHC2. Based on their relative proportions (MHC1:MHC2), the tissues fell into one of three groups: (1) 55:45 - rAo, rCA, dFA; (2) 60:40 - dSV, BAo, rGI; and (3) 65:35 - rUt, rFA. Group 1 differed significantly from group 3 in the proportion of each MHC. One dimensional peptide maps indicated that BAo, dSV and dFA were similar while subtle differences existed between rUt and rAo. Differences between rUt and rAo were also observed in their cross-reactivity to a monoclonal antibody to smooth muscle MHC, confirming the differences seen on peptide maps. These results indicate that there are intertissue and inter- and intraspecies differences in smooth muscle MHCs. The significance of these differences to muscle function remains to be determined.     

Effect of atriopeptin on the adrenergic mechanism regulating blood vessel tonus

The direct action of atriopeptin on the cell regulation mechanism of the smooth muscle in isolated segments of the portal vein, aorta, pancreatic and cerebral arteries have been studied. It was found that atriopeptin induce the direct relaxation of the smooth muscle in the main vessels only (aorta, portal vein). In the cerebral and pancreatic arteries atriopeptin stops norepinephrine-induced contractions. The data obtained show that the action of the atriopeptin is mediated by Na+-K+ pump activation of smooth muscle cells and restricts vasoconstriction of catecholamine effect.     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

SARCOPLASMIC RETICULUM AND EXCITATION-CONTRACTION COUPLING IN MAMMALIAN SMOOTH MUSCLES

The sarcoplasmic reticulum (SR) was studied in the smooth muscles of rabbit main pulmonary artery, mesenteric vein, aorta, mesenteric artery, taenia coli, guinea pig mesenteric artery, and human uterus, and correlated with contractions of the smooth muscles in Ca-free media. SR volumes were determined in main pulmonary artery (5.1%), aorta (5%), portal-anterior mesenteric vein (2.2%), taenia coli (2%), and mesenteric artery (1.8%): because of tangentially sectioned membranes these estimates are subject to a correction factor of up to +50% of the values measured. Smooth muscles that contained a relatively large volume of SR maintained significant contractile responses to drugs in the virtual absence of extracellular calcium at room temperatures, while smooth muscles that had less SR did not. The unequal maximal contractions of main pulmonary artery elicited by different drugs were also observed in Ca-free, high potassium-depolarizing solution, indicating that they were secondary to some mechanism independent of changes in membrane potential or calcium influx. Longitudinal tubules of SR run between and are fenestrated about groups of surface vesicles separated from each other by intervening dense bodies. Extracellular markers (ferritin and lanthanum) entered the surface vesicles, but not the SR. The peripheral SR formed couplings with the surface membrane: the two membranes were separated by gaps of approximately 10 nm traversed by electron-opaque connections suggestive of a periodicity of approximately 20–25 nm. These couplings are considered to be the probable sites of electromechanical coupling in twitch smooth muscles. Close contacts between the SR and the surface vesicles may have a similar function, or represent sites of calcium extrusion. The presence of both thick and thin myofilaments and of rough SR in smooth muscles supports the dual, contractile and morphogenetic, function of smooth muscle.     

New agonist and antagonist analogues of bradykinin

Three new analogues of bradykinin (BK) have been tested for their agonistic and antagonistic actions on the rabbit jugular vein and the guinea pig ileum (B2 receptors), and six were studied on rabbit aorta strips (B1 receptors). Substitution of Gly4, Phe5, and Phe8 in BK with D-Trp gives analogues with a relative affinity lower than 1.0% as compared with BK. These analogues have no antagonistic properties on the rabbit jugular vein and on guinea pig ileum (B2 receptors). Substitution of Pro7 in des-Arg9-BK by Gly and by D-Ala give compounds that antagonise the effects of kinins on the rabbit aorta strips (B1-receptor system). These new antagonists are fairly potent with a pA2 value of 6.03 to 7.29 and seem competitive because the pA2--pA10 values approximate 0.95. These results suggest that the orientation of Phe8 is critical for the activation of B1 receptors by kinins.     

Different accessibility from the artery and the portal vein of alpha- and beta-receptors involved in the sympathetic nerve action on glycogenolysis and hemodynamics in perfused rat liver

In perfused rat liver perivascular nerve stimulation (7.5 Hz, 20 V, 2 ms, 5 min) at the liver hilus caused an increase in glucose and lactate output and a decrease in flow. The influence of the alpha 1-receptor blocker prazosine and the beta-blocker propranolol on these nerve effects was studied in the isolated rat liver perfused classically via the portal vein only and, as developed recently, via both the hepatic artery and the portal vein. 1) In livers perfused via the portal vein only the nerve stimulation-dependent metabolic alterations were nearly completely inhibited by prazosine (5 microM), but not influenced by propranolol (10 microM). The hemodynamic changes were lowered to only 33% by prazosine and not altered by propranolol either. 2) In livers perfused via the hepatic artery (100 mm Hg, 20-40% of flow) and the portal vein (10 mm Hg, 80-60% of flow)--similar to portal perfusions--the nerve stimulation--dependent metabolic alterations were almost completely blocked by arterial, portal or simultaneously applied arterial and portal prazosine. However--in contrast to portal perfusions--the metabolic alterations were reduced to about 20% (glucose) and 50% (lactate) also by propranolol independently of its site of application. The decrease in flow was reduced by prazosine to about 60%, 50% and 30% when applied via the artery, the portal vein or via both vessels, respectively. The hemodynamic alterations were not influenced by propranolol. These results allow the following conclusions: A subpopulation of beta-receptors can play a permissive role in the alpha 1-receptor-mediated sympathetic nerve action on glucose and lactate metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of methionine-enkephalin on the spontaneous electrical and mechanical activity of the smooth muscle of the rat portal vein

In the longitudinal smooth muscle of the isolated rat portal vein, methionine-enkephalin (Met-enkephalin) increased the spontaneous contraction with a concentration as low as 10(-8)M. When the membrane activity was recorded using a microelectrode, Met-enkephalin enhanced the spike burst activity but without any effect on the resting membrane potential. Naloxone, phentolamine, atropine and reserpine pre-treatment did not inhibit the excitatory effect of Met-enkephalin on the spontaneous contraction. These results suggest that the excitatory effect of Met-enkephalin on the mechanisms involved in the automaticity may be a direct action on smooth muscle or relate to presynaptic action on a non-adrenergic non-cholinergic system.     

Distribution of the vasoconstrictor and vasorelaxant effects of norbormide along the vascular tree of the rat

Norbormide is a vasoconstrictor of rat peripheral arteries and a relaxant in rat aorta. To characterise norbormide actions within the rat vascular tree we have investigated its effects on the contractile function of rings from several arteries and veins. A maximal norbormide concentration (50 microM) failed to contract thoracic aorta and carotid artery, whereas in pulmonary artery, abdominal aorta, iliac, caudal, and femoral arteries it induced a contractile effect that was respectively 4.8 +/- 0.6, 18.4 +/- 1.5, 39 +/- 5, 144 +/- 7, and 260 +/- 22% of that induced by 90 mM KCl. In pulmonary, carotid, and iliac arteries, and in thoracic and abdominal aorta, 50 microM norbormide inhibited KCl-induced responses. Norbormide (50 microM) contracted all veins investigated. The effect, expressed as % of KCl-induced contraction, was 121 +/- 25, 154 +/- 14.5, 154 +/- 18.2, 203 +/- 19, and 267 +/- 33 for pulmonary vein, thoracic and abdominal vena cava, iliac and jugular veins, respectively. In jugular vein, as previously shown in rat caudal artery, norbormide contraction was abolished in Ca2+-free medium, was unaffected by the Ca2+ channel blocker nifedipine, and was relaxed by SK&F 96365, a blocker of store-operated Ca2+ channels. In conclusion: i) rat veins represent the main target for contractile norbormide action; ii) in both artery and veins norbormide contractions are generally inversely related to the calibre of the vessel; iii) norbormide-induced contraction is mediated by the same mechanism/s in arteries and veins; iiii) in norbormide-contracted arteries the drug activates both contractile and relaxing mechanisms.     

Does zaltoprofen antagonize the bradykinin receptors?

Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B₂ receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B₂ receptor antagonists (contractility, human isolated umbilical vein) or B₁ receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg⁹-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg⁹-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kinin-stimulated phospholipase A₂ activity in HEK 293 cells expressing recombinant forms of the rabbit B₁ or B₂ receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems.     

Cardiovascular actions of kinins in the rabbit.

The hypotensive action of bradykinin (BK) and congeners was measured in anesthetized rabbits by administering the peptides intravenously and intraarterially in order to evaluate their pulmonary inactivation. A systematic study of the distribution of receptors for BK in the cardiovascular system of the rabbit was approached: (a) by measuring the myotropic effects of several peptides related to BK in strips of large arteries and veins; (b) by recording the changes of tension and rate of isolated atria; and (c) by evaluating the changes of perfusion pressure in isolated hearts, kidneys, and ears. This investigation was extended to strips of aortae of various mammals and to isolated atria of guinea pigs, for comparison. Receptors for BK were classified into two main types, B1 and B2, using the order of potency of these agonists [Tyr(Me)8]-BK, BK, and [des-Arg9]-BK, and an antagonist, specific and competitive for the B1 receptors, the octapeptide [Leu-OMe8,des-Arg9]-BK. The results obtained in this study indicate that the complex cardiovascular effect of BK in vivo may result from direct actions on vascular smooth muscles, presumably mediated by at least two types of receptors, as well as from the release of endogenous prostaglandins. BK and congeners exert a direct action on vascular smooth muscle by stimulating specific receptors both of the B1 type (in the aorta, the large arteries, and the mesenteric vein) and of the B2 type (in the jugular vein); and these vascular tissues provide useful preparations for pharmacological studies of bradykinins. Isolated organs perfused through their main arteries with physiological medium respond to BK by an increase of perfusion pressure (vasoconstriction in isolated ears and kidneys) or by a decrease (vasodilation in the rabbit heart). The vascular effects of BK in the heart and the kidney depend in part on the release of endogenous prostaglandins and on the activation of receptors that appear to be of the B2 type. Like other endogenous hypotensive agents, BK appears to reduce the tonus of the peripheral vessels, while contracting large arteries and veins. The results obtained in vitro are discussed with respect to the hypotensive effect in vivo and to the role of kinins in inflammation and oedema.     

Ketamine: evidence of tissue specific inhibition of neuronal and extraneuronal catecholamine uptake processes

Ketamine (1.1 X 10(-5) to 3.7 X 10(-4) M) potentiated catecholamine responses of rat anococcygeus muscle and rabbit aorta in vitro. In the anococcygeus, potentiation was abolished by cocaine (2.9 X 10(-5) M) pretreatment or by chemical sympathectomy using 6-hydroxydopamine (6-OHDA), but was unaffected by pretreatment with the extraneuronal uptake inhibitor cortisol (8.3 X 10(-5) M), or the catechol-O-methyltransferase inhibitor tropolone (2.4 X 10(-4) M). The action of ketamine mimicked the potentiating effect of cocaine on tyramine responses. In contrast, the potentiation by ketamine in rabbit aorta was unaffected by cocaine or 6-OHDA but was abolished by cortisol or tropolone; and ketamine potentiated tyramine responses, whereas cocaine inhibited them. Thus the mechanism of action by which ketamine produces potentiation of catecholamines in these two tissues is completely different. These results suggest that ketamine has the unusual ability to block neuronal and extraneuronal uptake and that the predominating mechanism will depend on the type of tissue examined and the morphology of its adrenergic innervation.     

Arterial wall neovascularization induced by glycerol

An intense and significant neovascularization, with numerous capillaries growing into the media layer of the rat femoral artery, was demonstrated when glycerol was administered into the interstitium between the femoral vein and the femoral artery. The maximum microvascularization was observed at days 7 and 9 after glycerol administration. Afterwards, involution of the majority of the newly-formed microvessels in the arterial wall occurred. Other substances containing glycerol in their molecules, such as triacetyl-glycerol and tributyril-glycerol, failed to produce significant neovascularization in the media layer of the femoral artery. Neovascularization of the arterial wall was preceded by a considerable decrease in the number of the smooth muscle cells, which experienced apoptosis and necrobiosis, disappearing in extense areas of the arterial segment affected by glycerol. Coinciding with neovascularization and microvascular involution, repopulation of the media layer by smooth muscle cells was observed.     

The smooth muscle cross-bridge cycle studied using sinusoidal length perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (, Pharmacol. Rev. 20:197-272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P(i)) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P(i), force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P(i) release. For the aorta, elevating P(i) decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P(i) state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased approximately 50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P(i) release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.     

Effects of endothelin and vasopressin on portal pressure of rats

Endothelin is a vasoconstrictor peptide which has recently been isolated and sequenced from the vascular endothelial cells. It was reported to increase blood pressure in vivo and produce a prolonged contraction with a slow onset in vitro. The purpose of this study was to investigate whether endothelin can lower the portal pressure as another endogenous vasoconstriction peptidevasopressin (AVP) can. Heart rate, systemic blood pressure, portal pressure, and portal vein blood flow were measured. Effects of endothelin on these parameters were compared with those of AVP. Endothelin 10(-10) mol/Kg significantly decreased all of the parameters mentioned. At the higher dose (5 x 10(-10) mol/Kg), however, the portal pressure and blood pressure were increased and portal vein blood flow was unchanged. On the other hand, AVP decreased the portal pressure and portal vein blood flow but elevated the systemic blood pressure. In vitro experiments revealed that endothelin contracted both tail artery and portal vein of rat and vasopressin contracted only tail artery. We concluded that although both are endogenous vasoconstricting peptides, endothelin and AVP affect differently on arterial and venous vascular beds as well as on portal pressure.     

Deformation-induced endothelin B receptor-mediated smooth muscle cell apoptosis is matrix-dependent

To maintain normal blood flow, pressure overload in both arteries and veins requires a structural adaptation of the vessel wall (remodelling) that involves smooth muscle cell (SMC) hypertrophy and/or hyperplasia. Due to its potent vasoconstrictor and growth-promoting effects, endothelin-1 (ET-1) is a likely candidate to initiate and/or promote remodelling in blood vessels exposed to a chronic increase in blood pressure. To test this hypothesis, isolated segments of the rabbit carotid artery and jugular vein were perfused at different levels of intraluminal pressure. In both types of segments, pressure overload (160 and 20 mmHg, respectively) resulted in an increase in endothelial prepro-ET-1 and SMC endothelin B receptor (ETB-R) expression. Moreover, in pressurised segments from the carotid artery an ETB-R antagonist-sensitive increase in SMC apoptosis in the media was observed, while in the vein medial SMC started to proliferate. Isolated SMC from these rabbit blood vessels as well as from the aorta and vena cava of the rat, when cultured on a collagen or laminin matrix, uniformly revealed an ETB-R-mediated increase in apoptosis upon exposure to mechanical deformation plus exogenous ET-1 (10 nmol/L). However, when grown on a fibronectin matrix, the cultured SMC did not respond with an increase in apoptosis under otherwise identical experimental conditions. These findings suggest that deformation-induced activation of the endothelin system in the vessel wall not only plays a crucial role in remodelling, but that the structural components of the vessel wall, in particular the cell-matrix interaction, determine how SMC respond phenotypically to these changes in gene expression.