Alzheimer's disease and its treatment-yesterday, today, and tomorrow.

Alois Alzheimer described the first patient with Alzheimer's disease (AD) in 1907 and today AD is the most frequently diagnosed of dementias. AD is a multi-factorial neurodegenerative disorder with familial, life style and comorbidity influences impacting a global population of more than 47 million with a projected escalation by 2050 to exceed 130 million. In the USA the AD demographic encompasses approximately six million individuals, expected to increase to surpass 13 million by 2050, and the antecedent phase of AD, recognized as mild cognitive impairment (MCI), involves nearly 12 million individuals. The economic outlay for the management of AD and AD-related cognitive decline is estimated at approximately 355 billion USD. In addition, the intensifying prevalence of AD cases in countries with modest to intermediate income countries further enhances the urgency for more therapeutically and cost-effective treatments and for improving the quality of life for patients and their families. This narrative review evaluates the pathophysiological basis of AD with an initial focus on the therapeutic efficacy and limitations of the existing drugs that provide symptomatic relief: acetylcholinesterase inhibitors (AChEI) donepezil, galantamine, rivastigmine, and the N-methyl-D-aspartate receptor (NMDA) receptor allosteric modulator, memantine. The hypothesis that amyloid-ß (Aß) and tau are appropriate targets for drugs and have the potential to halt the progress of AD is critically analyzed with a particular focus on clinical trial data with anti-Aß monoclonal antibodies (MABs), namely, aducanumab, lecanemab and donanemab. This review challenges the dogma that targeting Aß will benefit the majority of subjects with AD that the anti-Aß MABs are unlikely to be the "magic bullet". A comparison of the benefits and disadvantages of the different classes of drugs forms the basis for determining new directions for research and alternative drug targets that are undergoing pre-clinical and clinical assessments. In addition, we discuss and stress the importance of the treatment of the co-morbidities, including hypertension, diabetes, obesity and depression that are known to increase the risk of developing AD.

Defying the economists: a decrease in heart rate improves not only cardiac but also endothelial function.

Ivabradine has proven therapeutic efficacy for cardiac ischaemia and, until proved otherwise, is a very specific inhibitor of the cardiac sinoatrial node I(f) current. In the current issue of the British Journal of Pharmacology, Drouin et al. demonstrated that chronic treatment of the human apoB-100 transgene dyslipidaemic mouse with ivabradine significantly improved endothelium-dependent vasodilatation to ACh in renal and cerebral arteries and that the beneficial effects of ivabradine result secondarily to the lowering of heart rate. These data suggest that drugs that target the I(f) current have potential benefits not only as anti-ischaemics but also as agents for the treatment of endothelial dysfunction.

Metformin is not just an antihyperglycaemic drug but also has protective effects on the vascular endothelium.

Metformin, a synthetic dimethyl biguanide, has been in clinical use for over 55 years, and today is considered the first-choice drug for the treatment of type 2 diabetes used by an estimated 125 million people worldwide. Metformin is orally effective, not metabolized, excreted unchanged by the kidney, relatively free of side effects and well tolerated by the majority of patients. Of importance is that the United Kingdom Prospective Diabetes Study 20-year study of type 2 diabetics, completed in 1998, compared patients treated with insulin, sulfonylureas and metformin and concluded that metformin provided vascular protective actions. Cardiovascular disease is the primary basis for the high morbidity and mortality that is associated with diabetes and that metformin proved to be protective resulted in a dramatic increase in its use. The vascular protective actions of metformin are thought to be secondary to the antihyperglycaemic effects of metformin that are mediated via activation of AMP kinase and subsequent inhibition of hepatic gluconeogenesis, fatty acid oxidation as well as an insulin sensitizing action in striated muscle and adipose tissue. As reflected by a number of clinical studies, patients treated with metformin also have improvement in endothelial function as measured by the use of plethysmography and measurement of flow-mediated vasodilatation. These data as well as data from animal studies are supportive that metformin has a direct protective action on the vascular endothelium. In this review article, we discuss the pharmacology of metformin and critique the literature as to its cellular sites and mechanism(s) of action.

Role of NO in vascular smooth muscle and cardiac muscle function.

NO is a key transducer of a vasodilator message from the endothelium to vascular smooth muscle. Recently, its actions as a negative inotrope in cardiac muscle have been discovered. In the vasculature, it is synthesized under physiological conditions following activation of a low-output, Ca(2+)-dependent NO synthase (NOS) in endothelial cells. Immune activation triggers the expression of a high-output, Ca(2+)-independent NOS in the vasculature and myocardium, causing the overproduction of NO and significant cardiovascular dysfunction. In this article, Richard Schultz and Chris Triggle briefly review recent findings concerning the role of NO, and other endothelium-derived factors, in vascular smooth muscle function and consider the consequences of its production in the heart.

Endothelin receptor blockade improves endothelial function in human internal mammary arteries.

OBJECTIVE: Endothelial dysfunction, specifically endothelium-derived contracting factors have been implicated in the development of arterial conduit vasospasm. The potent vasoconstrictor endothelin-1 (ET-1) has received much attention in this regard. The present study was designed to evaluate the role of ET-1 in the development of endothelial dysfunction in human internal mammary arteries (IMA). To this aim, we examined the effects of specific and non-specific ET-receptor antagonists on endothelial function (assessed using acetylcholine (ACh)-induced vasodilation) in segments of IMA obtained during coronary artery bypass graft (CABG) surgery. METHODS: Vascular segments of IMA were obtained from 51 patients undergoing elective coronary artery bypass graft (CABG) surgery and in vitro endothelium-dependent and -independent responses to ACh and sodium nitroprusside (SNP) were assessed. Isometric dose response curves (DRC) to ACh and SNP were constructed in pre-contracted rings in the presence and absence of bosentan (ET(A/B) receptor antagonist, 3 microM), BQ-123 (ET(A) antagonist, 1 microM) and BQ-788 (ET(B) antagonist, 1 microM) using the isolated organ bath apparatus. Percent maximum relaxation (%E(max)) and sensitivity (pEC(50)) were compared between interventions. RESULTS: ACh caused dose-dependent endothelium-mediated relaxation in IMA (%E(max) 43+/-4, pEC(50) 6. 74+/-0.12). In the presence of bosentan, BQ-123 and BQ-788 ACh-induced relaxation was significantly augmented (%E(max) bosentan 60+/-3, BQ-123 56+/-4, BQ-788 53+/-5 vs. control 43+/-4, P<0.05) without affecting sensitivity. The effects of these antagonists were endothelium-specific since endothelium-independent responses to SNP remained unaltered. Furthermore, the beneficial effects were independently and maximally mediated by ET(A) and ET(B) receptors (%E(max) BQ-123 56+/-4 vs. BQ-788 53+/-5 vs. bosentan 60+/-3, P>0. 05). CONCLUSIONS: These data uncover, for the first time, beneficial effects of ET receptor blockade on endothelial-dependent vasorelaxation in human IMA.

Characterization of a specific, high affinity [3H]arginine8 vasopressin-binding site on liver microsomes from different strains of rat and the role of magnesium.

A single class of high affinity, low capacity, specific binding sites for [3H]arginine8 vasopressin (AVP) has been characterized in a plasma membrane-enriched microsomal fraction of the rat liver. Specific binding was saturable, linear with protein concentration, reversible, and 40-65% of the total binding. Binding at 25 C achieved a plateau after 30 min of incubation, whereas at 4 C, equilibrium was reached more slowly, and the level of binding was reduced. The presence of magnesium (Mg2+) in the assay medium enhanced the affinity of specific binding, while calcium and higher levels of sodium and potassium decreased binding. Scatchard analysis of binding in the presence of Mg2+ (5 mM) revealed an apparent mean +/- SE equilibrium dissociation constant (Kd) of 0.29 +/- 0.08 nM, with a maximal site density (Bmax) of 150.4 +/- 25.0 fmol/mg; in contrast, the Kd was 1.93 +/- 0.33 nM and the Bmax was 113.4 +/- 40.0 fmol/mg in the absence of Mg2+. No significant differences in Kd and Bmax were observed among membrane fractions derived from spontaneously hypertensive rats, Wistar-Kyoto rats, Long-Evans rats, and Brattleboro rats. Plasma levels of AVP were similar in spontaneously hypertensive, Wistar-Kyoto, and Long-Evans rats, but AVP was not detectable in the plasma from DI rats. Competitive inhibition of specific [3H]AVP binding by unlabeled AVP and related peptides showed the following Ki values: AVP, 0.19 nM; LVP, 1.7 nM; oxytocin, 41.4 nM; desamino AVP, 0.38 nM; [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid) 4-Val,8-D-Arg] VP2-(O-methyl)tyrosine]AVP, 1.8 nM; desglycinamide AVP, 2.2 microM. The neuropeptide metabolite of AVP [[pGlu4,Cyt6] AVP-(4-9)], angiotensin II, and other unrelated peptides did not displace [3H]AVP, demonstrating the specificity of AVP and its related biologically active peptides for this binding site. Moreover, the rank order of potency for displacement of [3H]AVP binding by these various peptides parallels their reported glycogenolytic activity in liver and/or their agonistic or antagonistic potency in vascular smooth muscle. Finally, the Mg2+-induced increase in the affinity of [3H]AVP for this liver binding site is similar to the reported effect of Mg2+ on the contractile responses of vascular smooth muscle to AVP (i.e. increased affinity). The results are consistent with the interpretation that the high affinity receptor site characterized in rat liver microsomes is of the V1 type.

Hepatic vasopressin receptor: differential effects of divalent cations, guanine nucleotides, and N-ethylmaleimide on agonist and antagonist interactions with the V1 subtype receptor.

Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin [( 3H]AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ greater than Mn2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ greater than Co2+ greater than Ca2+ greater than Mg2+ greater than Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from approximately 2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2-specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTP gamma S and GDP beta S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTP gamma S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Prevention of hypertension and vascular changes by captopril treatment.

Treatment of female spontaneously hypertensive rats (SHR) and control Wistar-Kyoto (WKY) rats with captopril was carried out by the addition of the drug in the drinking water throughout pregnancy and lactation and after weaning. At 28 weeks of age, average systolic blood pressure of treated SHR was 113 +/- 3 mm Hg, which was below that of control SHR (188 +/- 3 mm Hg) and WKY rats (124 +/- 3 mm Hg). Body weight and heart rate of the SHR were not affected by the treatment. Tissue level of catecholamines was increased by captopril treatment in the superior cervical ganglia but remained unchanged in the plasma, heart, mesenteric arteries, and the adrenal glands of both SHR and WKY rats. Left ventricular weight, wall thickness, and internal diameter of the left ventricle in the SHR were reduced by the treatment. Morphometric measurements of the mesenteric arteries showed that vascular alterations present in the control SHR were prevented by the treatment. In the superior mesenteric artery and large mesenteric artery, smaller lumen size at maximal relaxation found in the control SHR was normalized to the level of the WKY rats. Hypertrophy of the medial wall in the superior mesenteric, large and small mesenteric arteries, and an increase in the number of smooth muscle cell layers in the large mesenteric artery of the SHR were prevented by the treatment. Perfusion study of the mesenteric vascular bed showed that reactivity of these vessels to norepinephrine was reduced, and sensitivity to norepinephrine (as determined by the effective dose that causes 50% of maximal response) was increased in the SHR by captopril treatment. Sensitivity of the tail artery in response to norepinephrine was not altered by the treatment. We conclude that long-term treatment with captopril of SHR before and after birth prevented the development of hypertension, structural and functional alterations of the mesenteric arteries, and cardiac hypertrophy.

Structural and functional consequence of neonatal sympathectomy on the blood vessels of spontaneously hypertensive rats.

Neonatal sympathectomy of spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) was performed by a combined treatment with antiserum to nerve growth factor and guanethidine during the first 4 weeks after birth. The development of hypertension was completely prevented in the treated SHR: at 28 to 30 weeks of age, systolic blood pressure of treated SHR was 139 +/- 2 mm Hg as compared with 195 +/- 8 mm Hg in untreated SHR. The extent of sympathectomy was verified by histofluorescence. Fluorescence histochemistry for catecholamine-containing nerves showed a complete absence of adrenergic nerves in the mesenteric arteries of treated rats. A supersensitivity to norepinephrine was exhibited by mesenteric arteries, anococcygeus muscle, and tail arteries from the treated SHR and WKY. In the mesenteric vascular bed, maximal response to norepinephrine was significantly reduced by sympathectomy. Sympathectomy also abolished the responses (e.g., generation of excitatory junctional potentials) of tail arteries to electrical stimulation of perivascular nerves. Morphometric measurements of three categories of mesenteric arteries showed that sympathectomy had no effect on the hypertrophic change of smooth muscle cells in the conducting vessels, but it prevented the hyperplastic changes of the muscle cells from reactive, muscular arteries and small resistance vessels. These results suggest that one of the primary roles of the overactive sympathetic nervous system in the development of hypertension in SHR is manifested through its trophic effect on the arteries of SHR. This trophic effect appears to cause a hyperplastic change in the smooth muscle cells in the reactive and resistance vessels, thereby contributing to the development of hypertension in older SHR.

Pressor actions of arginine vasopressin in pithed Sprague-Dawley, Wistar-Kyoto and spontaneously hypertensive rats before and after treatment with nifedipine or pertussis toxin.

The pressor actions of arginine vasopressin (AVP) were examined in pithed Sprague-Dawley and Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Prior to pithing, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded via an intra-arterial catheter from sodium pentobarbital anaesthetized rats. SBP and DBP recorded from SHR were significantly greater than those from Sprague-Dawley and WKY rats. However, after pithing, there were no significant differences between DBP among the various strains. Pertussis toxin pretreatment significantly reduced the prepithing SBP and DBP of the SHR but not Sprague-Dawley or WKY rats. Administration of nifedipine significantly reduced DBP of pithed rats. The dose-diastolic pressure response curves obtained from infusion of AVP in Sprague-Dawley and WKY rats were not significantly different from one another, but the maximal vasopressor responses to AVP in pithed SHR were enhanced. Administration of nifedipine to Sprague-Dawley and WKY rats did not affect the dose-response curve to AVP, but nifedipine administration in SHR led to a significant inhibition of the pressor responses to AVP. Furthermore, pertussis toxin pretreatment of rats significantly reduced a component of the AVP pressor effect in SHR but not Sprague-Dawley or WKY rats. We speculate that, in SHR, vasopressin receptors are coupled to a pertussis toxin-sensitive G protein that, in turn, may couple to a dihydropyridine-sensitive calcium channel and also to a pertussis-insensitive G protein that is probably coupled to the phospholipase C/intracellular calcium release process. A component of the elevated blood pressure in SHR is also regulated by a pertussis toxin-sensitive process.(ABSTRACT TRUNCATED AT 250 WORDS)

Subcellular membrane properties in vascular and non-vascular smooth muscles of Dahl hypertensive rats.

Subcellular membrane fractions were isolated from mesenteric arteries and vas deferens of salt-resistant and salt-sensitive Dahl rats on low-salt (0.4% NaCl) high-salt (8.0% NaCl) diets. Only the salt-sensitive Dahl rats on the high-salt diet developed sustained high blood pressure (BP) after 5 weeks of the high-salt diet. Protein contents, membrane associated enzyme activities, calcium ion (Ca2+) binding and ATP-dependent CA2+ transport were compared in fractions isolated from all four groups. No obvious changes were observed, except for minor enhancement in magnesium ion (Mg2+)- and CA2+ ATPase activities of mesenteric arterial membranes isolated from salt-sensitive Dahl rats on high-salt diet compared to those from other groups of rats. The membrane fractions from vas deferens of salt-sensitive Dahl rats on the high-salt diet, on the other hand, showed decreased ATP-dependent Ca2+ transport compared to those from salt-sensitive Dahl rats on the low-salt diet. No difference was observed in membrane fractions isolated from salt-resistant Dahl rats on high-salt diet compared to those on low-salt diet. The significance of these observations are discussed in relation to the findings previously obtained from corresponding smooth muscle tissues of spontaneous hypertensive rats (SHR).

Cardiac regression and blood pressure control in the Dahl rat treated with either enalapril maleate (MK 421, an angiotensin converting enzyme inhibitor) or hydrochlorothiazide.

Enalapril maleate (MK 421), and hydrochlorothiazide were used to evaluate the control of hypertension and reversal of myocardial hypertrophy in Dahl sensitive (DS) and Dahl resistant (DR) rats given either a high (8% NaCl) or a low salt (0.4% NaCl) diet. Groups of six-week-old male DS and DR rats were treated with enalapril (15-100 mg/kg/day) in drinking water for eight weeks. Additional comparable groups of DS and DR were also treated with hydrochlorothiazide (60-400 mg/kg/day). Systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR) and heart weight/body weight (Hwt/Bwt) ratio were determined. We observed significant reduction in Hwt/Bwt ratio (P less than 0.001) along with control of SBP and DBP in the DS given a high salt diet treated with either enalapril or hydrochlorothiazide. However, in the DR given a high salt diet, cardiac regression (Hwt/Bwt ratio, P less than 0.05), SBP and DBP (P less than 0.01) reduction were seen only with enalapril. Similarly, cardiac regression (Hwt/Bwt ratio, P less than 0.05) was observed along with reduction of SBP and DBP (P less than 0.001) in the DS given a low salt diet and DR given enalapril. These data indicate that enalapril reduced SBP and DBP in association with cardiac regression in hypertensive and normotensive rats. In contrast, hydrochlorothiazide only reduced SBP, DBP and caused cardiac reversal (Hwt/Bwt ratio) in DS placed on a high salt diet.

Synthesis and calcium channel antagonist activity of dialkyl 4- (dihydropyridinyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinecarboxylates.

The sodium borohydride reduction of 3,5-disubstituted 1,4-dihydro-2,6-dimethyl-4-(pyridinyl)pyridines 2 and 5 in the presence of methyl, phenyl, or tert-butyl chloroformate afforded the respective 4-(dihydropyridinyl)-1,4-dihydropyridines 4 and 6 in good yield. Products 4 comprised a mixture of the 1,2- and 1,6-dihydropyridinyl regioisomers 4a and 4b where 4a was always the predominant regioisomer. Compounds possessing a 4-[dihydro-1-(phenoxycarbonyl)-3-pyridinyl] substituent, such as 26, were also a mixture of two regioisomers 26a and 26b, and each regioisomer existed as a mixture of two rotamers in Me2SO-d6 at 25 degrees C (26a', 26a'', and 26b', 26b'') due to restricted rotation about the nitrogen-to-carbonyl carbamate bond. The calcium antagonist activities for 4 and 6 were determined by using the muscarinic receptor-mediated Ca2+-dependent contraction of guinea pig ileal longitudinal smooth muscle. The relative order of activities for the 4-(dihydropyridinyl) analogues was 4-(dihydro-3-pyridinyl) greater than 4-(dihydro-4-pyridinyl). Increasing the size of the C-3(5) alkyl ester substituents increased activity. Compounds having nonidentical ester substituents were more active than those having identical ester substituents. Replacement of the C-3 and/or C-5 ester substituents by a cyano substituent(s) decreased activity significantly. An approximate 1:1 correlation between the IC50 value for inhibition of [3H]nitrendipine binding and inhibition of the tonic component of the muscarinic-induced contractile response was observed. The test results suggest that a 4-(dihydropyridinyl) substituent is bioisosteric with a 4-(nitrophenyl) substituent on a 1,4-dihydropyridine ring where m- and p-nitrophenyl are bioisosteric with the 4-[1,2(1,6)-dihydro-3-pyridinyl] 4 and 4-(1,2-dihydro-4-pyridinyl) 6 isomers, respectively.

Synthesis and calcium channel antagonist activity of dialkyl 1,4-dihydro-2,6-dimethyl-4-(pyridinyl)-3,5-pyridinedicarboxylates.

The Hantzsch condensation of alkyl acetoacetates 3 with methyl 3-aminocrotonate (4) and pyridinecarboxaldehydes 5 afforded the unsymmetrical alkyl methyl 1,4-dihydro-2,6-dimethyl-4-(pyridinyl)-3,5-pyridinedicarboxylates 6, whereas condensation of 3 with 5 and ammonium hydroxide gave the symmetrical dialkyl 1,4-dihydro-2,6-dimethyl-4-(pyridinyl)-3,5-pyridinedicarboxylates 7. The calcium channel antagonist activities of disubstituted 1,4-dihydro-3,5-pyridinedicarboxylates 6,7, and 9 were determined with use of the muscarinic-receptor-mediated Ca2+-dependent contraction of guinea pig ileal longitudinal smooth muscle. The relative potency order for isomeric pyridinyl analogues 6 and 7 was 2-pyridinyl greater than 3-pyridinyl greater than 4-pyridinyl. Increasing the size of the alkyl ester substituents enhanced activity. Compounds having nonidentical ester substituents were more potent than those having identical ester substituents. Replacement of the C-3 and/or C-5 ester substituent(s) by a cyano substituent(s) decreased activity significantly. An approximate 1:1 correlation between the IC50 value for inhibition of [3H]nitrendipine binding and inhibition of the tonic component of the muscarinic-induced contractile response was observed. The test results suggest that a 4-(pyridinyl) substituent is bioisosteric with a 4-(nitrophenyl) substituent on a 1,4-dihydropyridine ring system where o-, m-, and p-nitrophenyl are bioisosteric with 2-pyridinyl, 3-pyridinyl, and 4-pyridinyl, respectively.

Interactions of nitric oxide synthase inhibitors and dexamethasone with alpha-adrenoceptor-mediated responses in rat aorta.

1. The effects of NG-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), their D-isomers, and dexamethasone on noradrenaline (NA)-induced contractions and antagonism by alpha-adrenoceptor antagonists, have been investigated in rat isolated thoracic aortic rings with/without endothelium. 2. NA produced concentration-dependent contractions of isolated aortic rings with EC50 values of 2.41 +/- 0.54 (n = 21) and 28.00 +/- 8.50 (n = 25) nM for endothelium-denuded and -intact preparations respectively. Acetylcholine (ACh) relaxed NA-precontracted rings with intact, but not those denuded of endothelium. 3. Treatment with L-NAME (1-30 microM), or L-NMMA (10-500 microM), but not their D-isomers, resulted in an endothelium-dependent enhancement of NA-induced contractions. Pre-treatment, in vitro, with 0.5 microM dexamethasone neither directly potentiated, nor influenced L-NAME-induced potentiation of NA-mediated contractions in endothelium-intact rings; however, dexamethasone pretreatment reduced EC50 values for NA, and also prevented L-NAME-induced potentiation, in denuded rings equilibrated for 5 h under resting tension. 4. In both intact and denuded rings, phentolamine, prazosin and WB 4101 shifted NA concentration-response curves to the right; L-NAME, and also L-NMMA, but not their D-isomers, reversed the blockade as indicated by significant decreases in NA dose-ratios. In denuded rings, reversal by L-NAME or L-NMMA was prevented following pretreatment with dexamethasone. 5. Following treatment with 5 or 50 nM phenoxybenzamine (PBZ), NA concentration-response (C-R)curves were shifted to the right with marked depression of maximal responses; 100 microM L-NAME reversed the antagonism in both endothelium intact and denuded rings. However, 500 nM PBZ treatment resulted in complete abolition of the responses to NA, and contractions were not restored by either L-NAME or L-NMMA.6. 5-Hydroxytryptamine (5-HT)-induced contractions of aortic rings were potentiated by endothelium denudation and also by L-, but not D-, NAME. 5-HT-induced contractions were non-competitively antagonized by 10nM ritanserin, and 100 microM L-NAME partially reversed the antagonism in intact but not denuded rings.7. It is concluded that the inhibition of constitutive endothelial NO synthase and inducible smooth muscle NO synthase accounts for the ability of L-NAME, and L-NMMA, to potentiate the effects of agonists and reduce alpha-adrenoceptor antagonism in endothelium-intact and denuded rings. Furthermore,endothelial cell removal/damage triggers the induction of a smooth muscle NO synthase.

Lack of involvement of endothelin-1 in angiotensin II-induced contraction of the isolated rat tail artery.

1. The contribution of endothelin-1 (ET-1) to angiotensin II (Ang II)-mediated contraction of the isolated rat tail artery was assessed with measurements of tension, and cytosolic calcium ([Ca(2+)](i)). The distribution of the AT(1) receptor was studied with RT - PCR and immunohistochemistry. 2. Ang II induced an endothelium-independent contraction (pEC(50) 7.95+/-0.06 and E(max): 0.46 g+/-0.05 with endothelium vs 7.81+/-0.02 and 0.41 g+/-0.07 without endothelium; P>0.05). Ang II (0.003 - 0.3 microM)-induced a non-sustained contraction of endothelium-intact preparations which was not antagonized by BQ-123 (1 microM), but was inhibited by losartan (10 nM). In addition, the maximal contraction induced by ET-1 (0.1 microM) could be further increased by the addition of 0.1 microM Ang II. 3. Ang II (0.001 - 0.3 microM) elevated [Ca(2+)](i) in single vascular smooth muscle cells (VSMCs) in a dose-dependent manner (pEC(50) 9.12+/-0.26) and the Ang II-induced increases in [Ca(2+)](i) were not affected by a Ca(2+)-free solution, but were abolished by pretreatment with caffeine (5 mM). Ang II did not increase [Ca(2+)](i) in endothelial cells. ET-1 (0.1 microM) increased [Ca(2+)](i) in single VSMCs in a normal Ca(2+) containing physiological saline solution (PSS), but not in a Ca(2+)-free solution. 4. Ang II-induced contraction was insensitive to inhibition by nifedipine (0.1 microM), an antagonist of L-type voltage-gated Ca(2+) channels, and SK&F96365 (10 microM), which blocks non-selective cation channels, whereas that to ET-1 was inhibited by SK&F69365. 5. RT - PCR data indicate the expression of AT(1A) and AT(1B) on both VSMCs and endothelial cells, but immunohistochemical evidence illustrates that the AT(1) is located primarily on VSMCs. 6. These results indicate that endothelium-derived ET-1 is not involved in the Ang II-mediated vasoconstriction of the rat tail artery and that Ang II- and ET-1-mediated VSM contractions utilize distinct pathways.

Inhibition of field stimulation-evoked relaxations in rat oesophageal smooth muscle by the calcium antagonist PN 200-110.

1. The inhibitory effects of the 1,4-dihydropyridine calcium channel antagonist, PN 200-110 (isradipine), on field stimulation-evoked tetrodotoxin (TTX)-sensitive and -insensitive relaxations were studied in rat oesophageal smooth muscle of the tunica muscularis mucosae. 2. The TTX-insensitive relaxation was inhibited by PN 200-110 in a stereoselective manner with the (+)-(S)-isomer displaying a 1000 fold greater inhibitory potency than the (--)-(R) isomer. A similar potency was noted for inhibition of high K+ -evoked contractions. 3. TTX-sensitive relaxations evoked by field stimulation and contractions elicited by the muscarinic cholinoceptor agonist, cis-2-methyl-4-dimethylamino-methyl-1,3-dioxolane methiodide (cisdioxolane) were considerably less sensitive to inhibition by PN 200-110, although, again, stereoselectivity for PN 200-110 was apparent. 4. Pretreatment with (+)-(S)-PN 200-110 resulted in a non-competitive displacement of the Ca2+ concentration-response curves obtained in the presence of either isotonic 50 mM KCl or cisdioxolane. The effect of K+ was 10 fold more sensitive than that of cis-dioxolane. 5. The potency rank orders for inhibition of TTX-insensitive field stimulation-evoked relaxations and K+ -mediated contractions in a series of calcium channel antagonists were closely correlated; (+)-(S)-PN 200-110 showing highest potency followed by nifedipine, verapamil, diltiazem, (--)-(R)-PN 200-110. 6. It is concluded that TTX-insensitive relaxations are dependent upon an influx of extracellular Ca2+ through potential-operated calcium channels.

Photosensitization of oesophageal smooth muscle by 3-NO2-1, 4-dihydropyridines: evidence for two cyclic GMP-dependent effector pathways.

1. Photoactivated mechanical responses that resulted from exposure to 3-NO2-1,4-dihydropyridines (3-NO2-DHP5) or NO-donors were examined in rat isolated oesophageal smooth muscle with a view to determining the role of calcium and cyclic GMP. 2. Isometric contractile force was recorded in preparations bathed in normal Tyrode or 110 mM K(+)-depolarizing solution. Exposure to (+)-PN 202791, (+/-)-Bay K 8644 and (-)-PN 2020791 or the photodegradable NO-donors, sodium nitroprusside (SNP), streptozotocin (STZ) and sodium nitrite photosensitized precontracted tunica muscularis mucosae preparations in a concentration-dependent fashion. Photosensitizing potency followed the order: (+/-)-PN 202791 > (+/-)-Bay K 8644 > (-)-PN 202791 > SNP > STZ > NaNO2. 3. A low amplitude, slow photorelaxation (slope: 1 mg s-1) was obtained with the L-channel antagonists (-)-PN 202791 and (+)-Bay K 4407. Photosensitization by the agonist enantiomers (+)-PN 202 791 and (-)-Bay K 5407, as well as racemic Bay K 8644, was mimicked by NO donors and showed at least three different components, consisting of (i) a fast relaxation (slope: 140 mg s-1), (ii) a fast "off-contraction', and (iii) a delayed slow relaxation. The fast components, but not the delayed slow relaxation, were abolished by blockade of L-type voltage-operated calcium channels, chelation of extracellular calcium and skinning of the plasmalemma, suggesting their mediation by a process linked to calcium entry through L-channels. 4. Both cyclopiazonic acid (3-30 microM) and ryanodine (30 microM) inhibited the fast response. This inhibition was accelerated in the presence of extracellular calcium and resembled that seen in tissues exposed to the calcium ionophore A 23187 (1 microM). In calcium depleted tissues, cyclopiazonic acid (3 microM) prevented restoration of the cis-dioxolane-induced contraction following re-exposure to a calcium containing high K+ buffer, but failed to inhibit the photoresponse. 5. Both the fast and slow relaxations were potentiated by zaprinast (10 microM) and inhibited by LY B3583 (10 microM). However, in calcium-depleted, calyculin A-precontracted preparations only the slow relaxation was evident. 6. The present results support the conclusion that: (i) functional L-channels are required for the expression of the fast components of the 3-NO2-DHP- or NO-donor-induced photoresponse, (ii) NO photorelease followed by activation of soluble guanylyl cyclase is responsible for the photosensitizing activity of 3-NO2-DHPs and (iii) regulation of the contractile proteins via cyclic GMP-dependent phosphorylation may underlie the slow relaxation.

The effects of perfusion rate and NG-nitro-L-arginine methyl ester on cirazoline- and KCl-induced responses in the perfused mesenteric arterial bed of rats.

1. The purpose of this study was to characterize the effect of NG-nitro-L-arginine methyl ester (L-NAME) on the perfusion rate/pressure relations, and on the pressor responses induced to cirazoline and KCl in isolated, perfused mesenteric arterial beds from normotensive and spontaneously hypertensive rats. 2. The basal perfusion pressure of arterial beds perfused with either physiological salt solution (PSS) or PSS containing 1% polyvinylpyrrolidone increased as the perfusion rate increased. L-NAME, in concentrations up to 100 microM, failed to alter the basal pressure regardless of the perfusion rate and viscosity; however, at 5 microM, it potentiated cirazoline-induced vasoconstriction at each of the perfusion rates. 3. L-NAME but not D-NAME caused a leftward shift of cirazoline concentration-response curves with a marked increase in the maximal response. The potentiating action of L-NAME was abolished in arterial beds perfused with a Ca(2+)-free physiological salt solution and also in beds denuded of endothelium by an infusion of distilled water for 5 min. 4. In endothelium-intact and -denuded preparations, L-NAME potentiated KCl pressor responses; the endothelium-independent potentiation of KCl pressor activity was stereospecific, time-independent and was not prevented by the presence of dexamethasone (0.5 microM) in the perfusion medium. However, L-NAME failed to potentiate vasoconstriction obtained to KCl in arterial beds denervated by cold storage (4-5 degrees C) for 2 days. 5. The absence of K+ in the perfusate did not inhibit the ability of L-NAME to potentiate alpha-adrenoceptor-mediated pressor responses, and nor did L-NAME inhibit KCl-induced vasodilatation in preconstricted arteries. It was thus concluded that L-NAME does not affect Na+/K(+)-ATPase activity. 6. No differences in the potentiating ability of L-NAME on either cirazoline- or KCl-mediated pressor responses were apparent between normotensive Sprague Dawley (SD), Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats.7. Our data thus provide evidence that: the presence of a vasoconstrictor is required for basal nitricoxide (NO) release in the mesenteric arterial bed from either normotensive or spontaneously hypertensive rats; L-NAME causes potentiation of cirazoline- and KCl-induced vasoconstriction respectively by inhibiting endothelial and neuronal NO synthase(s). Furthermore, our data indicate that NO synthase activity is not impaired in the mesenteric arterial bed of spontaneously hypertensive rats.

Comparison of the pharmacological properties of EDHF-mediated vasorelaxation in guinea-pig cerebral and mesenteric resistance vessels.

In the presence of L-NNA (100 microM), indomethacin (10 microM) and ODQ (10 microM), acetylcholine induced a concentration-dependent vasorelaxation of guinea-pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K(+), indicating the contribution of an endothelium-derived hyperpolarizing factor (EDHF). In cerebral arteries, charybdotoxin (ChTX; 0.1 microM) completely inhibited the indomethacin, L-NNA and ODQ-insensitive relaxation; iberiotoxin (IbTX, 0.1 microM), 4-aminopyridine (4-AP, 1 mM), or barium (30 microM) significantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 microM) had no affect in either the mesenteric or cerebral artery. Neither clotrimazole (1 microM) nor 7-ethoxyresorufin (3 microM) affected EDHF-mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF-mediated relaxations in the cerebral artery. AM404 (30 microM), a selective anandamide transport inhibitor, did not affect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin-sensitive, but SR 141816A-insensitive manner. Ouabain (100 microM) almost abolished EDHF-mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K(+) (5 - 20 mM) to precontracted guinea-pig cerebral or mesenteric artery induced further vasoconstriction. These data suggest that in the guinea-pig mesenteric and cerebral arteries different EDHFs mediate acetylcholine-induced relaxation, however, EDHF is unlikely to be mediated by K(+).