Martin Pfaffendorf

Voltage-operated calcium channels are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries.

The role of L-type voltage-operated Ca2+ channels (VOCs) in myogenic responsiveness was studied in cannulated rat mesenteric small arteries [mean diameter at 100 mm Hg and full dilation was 329 +/- 9 (SE) micrometer]. Twenty-six arteries were cannulated and pressurized. The luminal cross-sectional area of these vessels was monitored continuously. To test for myogenic responsiveness, pressure was raised stepwise from 20 to 60 and from 60 to 100 mm Hg. Pressure elevation enhanced the vascular tone, reflecting spontaneous myogenic responsiveness. Nifedipine (1 and 10 microM) suppressed spontaneous myogenic responses. The alpha1-adrenoceptor agonist phenylephrine (1 and 10 microM), when administered at 20 mm Hg, elicited constriction and vasomotion, and potentiated myogenic constriction to subsequent pressure elevation. Nifedipine (1 and 10 microM) also suppressed phenylephrine-potentiated myogenic responsiveness. Stimulation of VOCs with BAY K 8644 (10-300 nM) had no effect at 20 mm Hg, but augmented myogenic responsiveness. K+ (16-46 mM) caused concentration-dependent constrictions when administered at 20 mm Hg, and potentiated myogenic responsiveness when the pressure was raised from 20 to 60 mm Hg. Thus, any intervention that blocked the VOCs also blocked myogenic responses. Therefore, we conclude that VOCs are essential for the myogenic responsiveness of cannulated rat mesenteric small arteries.

Similarities and differences between calcium antagonists: pharmacological aspects

Characteristics of three different calcium antagonist groups: Most important calcium antagonists used to treat cardiovascular disease belong to one of three main groups, phenylalkylamines, dihydropyridines and benzothiazepines. The best known drug in each group is verapamil, nifedipine and diltiazem, respectively. Dihydropyridines are predominantly vasodilators, with little or no primary cardiac activity; the tachycardia . caused by these compounds is a reflex phenomenon. Verapamil and related drugs are also vasodilators, with an additional depressant effect on atrioventricular conduction, heart rate and contractility. Diltiazems pharmacodynamic profile and side effects may be considered as intermediate between those of the dihydropyridines and verapamil. Characteristics of new calcium antagonists: Several new calcium antagonists have been introduced in the last few years, virtually all dihydropyridines. Compared with the older generation of calcium antagonists these newer drugs tend to have (1) a longer duration of action; (2) some selectivity for a specific vascular bed, such as resistance, coronary, renal or cerebral vessels; (3) a potentially useful extra component, such as diuretic or anti-atherogenic activity. Newer calcium antagonists described in this review: The newer compounds briefly characterized in this review are amlodipine, felodipine, isradipine, lacidipine, nimodipine, nisoldipine and nitrendipine. New slow-release formulations are also discussed. There is a particular emphasis on lacidipine and its potential for cardiovascular drug therapy.

The interaction between methylene blue and the cholinergic system.

1 The inhibitory effects of methylene blue (MB) on different types of cholinesterases and [3H]‐N‐methylscopolamine ([3H]‐NMS) binding to muscarinic receptors were studied. 2 Human plasma from young healthy male volunteers, purified human pseudocholinesterase and purified bovine true acetylcholinesterase were incubated with acetylcholine and increasing concentrations of MB (0.1–100 μmol l−1) in the presence of the pH‐indicator m‐nitrophenol for 30 min at 25°C. The amount of acetic acid produced by the enzymatic hydrolysis of acetylcholine was determined photometrically. 3 Rat cardiac left ventricle homogenate was incubated with [3H]‐NMS and with increasing concentrations of MB (0.1 nmol l−1–100 μmol l−1) at 37°C for 20 min. The binding of [3H]‐NMS to the homogenate was quantified by a standard liquid scintillation technique. 4 MB inhibited the esterase activity of human plasma, human pseudocholinesterase and bovine acetylcholinesterase concentration‐dependently with IC50 values of 1.05±0.05 μmol l−1, 5.32±0.36 μmol l−1 and 0.42±0.09 μmol l−1, respectively. MB induced complete inhibition of the esterase activity of human plasma and human pseudocholinesterase, whereas bovine acetylcholinesterase was maximally inhibited by 73±3.3%. 5 MB was able to inhibit specific [3H]‐NMS binding to rat cardiac left ventricle homogenate completely with an IC50 value of 0.77±0.03 μmol l−1, which resulted in a Ki value for MB of 0.58±0.02 μmol l−1. 6 In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant affinity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L‐arginine‐NO‐pathway, in particular when muscarinic receptor stimulation is involved.

Comparative effects of angiotensin II and its degradation products angiotensin III and angiotensin IV in rat aorta

1 In the present study, the contractile effects of angiotensin III (AIII) and angiotensin IV (AIV) compared with those of angiotensin II (All) were determined in rat aortic ring preparations. 2 All three peptides caused concentration‐dependent contractions with similar maximal responses. AIII proved approximately 4 times less potent than All, whereas AIV was about 1000 times less active than AII 3 The selective AT1‐receptor antagonist, losartan (10–300 nM) caused parallel rightward shifts of the concentration‐response curves (CRC) for all three peptides. The Schild plot slopes for the effect of losartan on AIII curves were significantly lower than unity (P<0.05). The selective AT2‐receptor antagonist, PD123177 did not influence the CRCs for All and AIV. However, the AIII curves were moderately shifted leftward in the presence of PD123177 (0.1 and 1 μm). 4 Destruction of the endothelium or incubation with the NO‐synthesis inhibitor NG‐monomethyl‐l‐arginine acetate (l‐NMMA) (0.1 mm) significantly enhanced the contractile responses to all three peptides. 5 Tachyphylaxis was investigated by constructing a second CRC for all three peptides, after an interval of 1 h. The presence of endothelium significantly enhanced the development of tachyphylaxis to all three peptides. However, in endothelium‐denuded preparations, the Emax value of the second curve elicted by All was about 50%, compared with the first one, whereas for AIII and AIV Emax values were as high as 90% and 100%, respectively. 6 Our results indicate that both AIII and AIV are less potent but similarly efficacious vasoconstrictor agents compared with AII Their contractile effects are also mediated by AT1receptors and probably modulated by endothelium. Tachyphylaxis induced by AIII and AIV proved weaker than that for AII Tachyphylaxis appears to be enhanced by the presence of an intact endothelium.

Endothelium-dependent, nitric oxide-mediated inhibition of angiotensin II-induced contractions in rabbit aorta

The role of endothelium in angiotensin II-induced contractions of the rabbit aorta and the mechanism involved were investigated. Destruction of the endothelium significantly shifted the concentration-response curve for angiotensin II to the left in a non-parallel manner and enhanced the maximal response. The EC50 and Emax values obtained from the rings with and without functional endothelium were 2.44 +/- 0.13 x 10(-9) M, 4.50 +/- 0.45 g and 1.21 +/- 0.14 x 10(-9) M (n = 8, P < 0.05), 5.73 +/- 0.55 g (n = 8, P < 0.05), respectively. Indomethacin (10(-5) M) did not significantly alter the concentration-dependent response to angiotensin II in the presence of endothelium. Three inhibitors of nitric oxide synthase (NG-monomethyl-L-arginine; NG-nitro-L-arginine, and NG-nitro-L-arginine methyl ester) at 10(-4) M caused a similar endothelium-dependent potentiation of angiotensin II-induced contractions in the aortic rings with, but not in those without endothelium. These effects were reversed by L-arginine (3 x 10(-3) M) but not by D-arginine (3 x 10(-3) M). Angiotensin II in a concentration range of 10(-16) to 10(-6) M did not relax the endothelium-intact rings precontracted with phenylephrine (2 x 10(-7) M). In the presence of endothelium, the angiotensin II subtype 2 receptor antagonist, 1-[(4-amino-3-methylphenyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro- 1H- imidazol[4,5-C]pyridine-6-carboxylic acid (PD 123177), caused neither relaxation of the rings precontracted with phenylephrine nor alteration of the concentration-response curve for angiotensin II.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vivo Characterization of Muscarinic Receptor Subtypes That Mediate Vasodilatation in Patients With Essential Hypertension

Attenuated cholinergic vasodilatation has been suggested as an endothelium-related mechanism involved in essential hypertension. We investigated the role of muscarinic (M) receptor subtypes in the forearm resistance vasculature. In eight white men with essential hypertension and eight matched normotensive control subjects (age of both groups, 47 +/- 4 years; mean +/- SEM), we infused the nonselective agonist methacholine in the presence of saline and the antagonists atropine (nonselective), pirenzepine (M1-selective), and AF-DX 116 (M2-selective) into the brachial artery and measured forearm blood flow and forearm vascular resistance using venous occlusion plethysmography. Affinity constants (pKb values) were determined from calculated plasma concentrations of the infused compounds and EC50 values. Sodium nitroprusside was given as an endothelium-independent control, and minimal forearm vascular resistance after 10 minutes of ischemia was used as a marker of structural vascular changes. Hypertensive patients showed higher minimal forearm vascular resistance, indicating structural vascular changes. However, sodium nitro-prusside- and methacholine-induced vasodilatation was similar in both groups, with apparent EC50 values (log moles per liter; mean +/- SEM) of -7.32 +/- 0.13 and -7.51 +/- 0.21 in hypertensive patients and -7.37 +/- 0.13 and -7.45 +/- 0.02 in control subjects, respectively. Atropine, pirenzepine, and AF-DX 116 caused a shift to the right of the concentration-response curve of methacholine, with apparent pKb values of 8.63 +/- 0.08, 6.81 +/- 0.13, and 5.51 +/- 0.29 in hypertensive individuals and 8.62 +/- 0.10, 6.98 +/- 0.08, and 5.49 +/- 0.09 in control subjects, respectively. Again, there were no statistically significant differences in these pharmacological parameters between hypertensive patients and normotensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Function of cryopreserved arterial allografts under immunosuppressive protection with cyclosporine A

PURPOSE Cryopreserved arterial allografts may be used for arterial reconstructive procedures. In this experimental study cryopreserved arteries were used as autografts and as allografts with or without immunosuppression with cyclosporine A. METHODS In group A (three dogs, six bilateral grafts) cryopreserved carotid artery autografts were implanted. In groups B and C female mongrel dogs (three dogs and six bilateral grafts in each group) received cryopreserved male carotid artery allografts. Dogs in group C were treated with cyclosporine A (25 mg/kg/day). After 3 months of implantation patency was assessed by angiography. Contractile responses to KCl and phenylephrine (Phe) and the endothelium-dependent relaxation response to methacholine (Met) were examined in segments of the grafts after excision. Medial thickness was assessed semiquantitatively. The grafts were stained for sex chromatin analysis to determine the origin of cells in allografts. RESULTS Patency: group A, 100% (6 of 6), group B, 66.6% (4 of 6), and group C, 100% (6 of 6). Functional responses: before implantation, after thawing, 2.7 +/- 0.5 mN (KCl), 4.8 +/- 1.0 mN (Phe), and 0.0% +/- 0.0% (Met), group A, 36.9 +/- 10.6 mN (KCl), 31.5 +/- 14.4 mN (Phe), and 59.3% +/- 20.4% (Met), group B, 0 for all agents used, group C, 34.0 +/- 7.5 mN (KCl), 28.8 +/- 7.0 mN (Phe), and 46.2% +/- 3.2% (Met). Morphologic characteristics: the media of grafts in group B showed significant thinning (p < 0.05). Smooth-muscle cells in vessel walls of grafts in group C were of female origin. CONCLUSION Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing. After 3 months of implantation cryopreserved arterial autografts and allografts under immunosuppressive treatment with cyclosporine A showed 100% patency and return of functional responses resulting from repopulation of grafts by host cells.

Comparison of cholinergic vasodilator responses to acetylcholine and methacholine in the human forearm.

In order to study the contribution of the nitric oxide (NO)-pathway to cholinergic vasodilatation in the resistance vessels of the human forearm, we infused acetylcholine (ACh; 0.1 1000 ng/kg/min) or methacholine (MCh; 0.1 A 100 ng/kg/min) in the presence of saline, the NO-scavenger and guanylate cyclase inhibitor methylene blue (MB; 1000 ng/kg/min), or the NO-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 30 micrograms/kg/min) into the brachial artery of normotensive volunteers (n = 32), using venous occlusion plethysmography. We calculated the plasma concentrations of the infused compounds to obtain EC50-values (-log mol/l). ACh and MCh both caused concentration-dependent vasodilatation (EC50-values of 6.43 +/- 0.05 and 7.24 +/- 0.08, respectively). MB (13 mumol/l) did not change basal forearm blood flow (FBF) when administered alone, but it markedly potentiated the vasodilator response to ACh, shifting the concentration-response curve (CRC) leftwards by 1.5 log-step (p < 0.001). MB did not affect MCh-induced vasodilatation. L-NMMA (1 mmol/l) alone caused dose-dependent vasoconstriction that was subject to tachyphylaxis. In addition, L-NMMA caused a steepening of the slopes of the CRCs of ACh, and MCh L-NMMA attenuated the ACh-/MCh-induced vasodilator responses in the lowest concentration ranges (p < 0.05) only, but did not alter the response at higher concentrations. The 10-fold higher potency of MCh compared to ACh can be explained by the more rapid degradation of ACh by cholinesterases. The observation that high concentrations of L-NMMA only affect vasodilation mediated by low concentrations of ACh or MCh, suggests a second mechanism in cholinergic vasodilatation, such as a direct effect on smooth muscle cells or the release of a relaxing factor other than NO.

Effects of losartan on vasoconstrictor responses to angiotensin II in the forearm vascular bed of healthy volunteers

OBJECTIVES The angiotensin type 1 (AT1) receptor antagonist, losartan (orally administered), decreases vasoconstrictor effects of angiotensin II (Ang II). Oral losartan is converted into the active metabolite, Exp3174, which causes most of the antagonistic effects. Effects of losartan as such have not been studied after its intra-arterial administration in humans. Therefore, we investigated the effects of both intra-arterially and orally administered losartan on AT1-receptor-mediated vasoconstriction. METHODS Forearm vascular resistance (FVR) was determined by venous occlusion plethysmography in 24 healthy subjects. Ang II (0.01, 0.1, 1.0, and 10.0 ng/kg/min) was infused into the brachial artery, before and after losartan, administered intra-arterially (dose range 100-3000 ng/kg/min) or orally (50 mg once daily for 5 days). RESULTS Ang II concentration-dependently increased FVR (P < 0.05); tachyphylaxis did not occur. Losartan alone did not change FVR. Intra-arterially infused losartan dose-dependently inhibited Ang-II-induced vasoconstriction. At a concentration of 10(-8) M Ang II, losartan reduced FVR, as a percentage of baseline values, from 287 +/- 30 to 33 +/- 8% (mean +/- s.e.m.; P < 0.05). Orally given losartan reduced FVR from 297 +/- 40 to 73 +/- 19% (P < 0.05). CONCLUSIONS Losartan, intra-arterially administered, causes no effect on baseline vascular resistance, but markedly inhibits Ang-II-induced vasoconstriction in the human forearm vascular bed. Relatively high doses of intra-arterial losartan were required when compared to the antagonism by the orally administered drug. These data indicate that Ang-II-induced vasoconstriction is mediated by AT1-receptors, which are blocked by losartan. The more effective antagonism exerted by oral losartan is presumably explained by the formation of Exp3174. Endogenous Ang II does not contribute to baseline vascular tone in healthy, sodium-replete, subjects.

The direct effects of thyroid hormones on rat mesenteric resistance arteries

Summary— The direct relaxant effects of thyroid hormones on mesenteric resistance vessels were investigated using an isometric wire myograph. Both the L‐ and the D‐isomers of thyroxine (T4) and triiodothyronine (T3) were studied. In contrast with the long‐term effects of thyroid hormones, both T4 enantiomers proved more potent in inducing vascular relaxation than the two T3 enantiomers. The interaction between thyroid hormones and calcium‐induced contractions was studied. T4 concentration dependently inhibited the Ca2+ induced contractions, showing noncompetitive interaction. Furthermore, we investigated whether the endothelium was involved in the relaxant effect to L‐T4. The T4 induced relaxation proved impaired by prior incubation with the nitric oxide (NO) inhibitor N‐ω‐nitro‐L‐arginine methylester HCl (L‐NAME, 0.1 μM), indicating that T4 is able to stimulate the production of endothelium‐derived NO. L‐T4‐induced relaxation was enhanced by prior incubation with indomethacin (10 μM), whereas in endothelium‐denuded preparations an unaltered response was found. The present results indicate that L‐T4‐induced relaxation is established by an indirect effect via the endothelium and by a direct effect on vascular smooth muscle cells, possibly by influencing calcium fluxes. Because vascular relaxation is established at supraphysiologic concentrations (approximately 100 times the basal level) of thyroid hormone, it is concluded that the direct effect of thyroid hormone on mesenteric vascular smooth muscle cells are not relevant for the in vivo situation.