Hydrogen sulfide and sulfaceutic or sulfanutraceutic agents: Classification, differences and relevance in preclinical and clinical studies.

Hydrogen sulfide (H2S) has been extensively studied as a signal molecule in the body for the past 30 years. Researchers have conducted studies using both natural and synthetic sources of H2S, known as H2S donors, which have different characteristics in terms of how they release H2S. These donors can be inorganic salts or have various organic structures. In recent years, certain types of sulfur compounds found naturally in foods have been characterized as H2S donors and explored for their potential health benefits. These compounds are referred to as "sulfanutraceuticals," a term that combines "nutrition" and "pharmaceutical". It is used to describe products derived from food sources that offer additional health advantages. By introducing the terms "sulfaceuticals" and "sulfanutraceuticals," we categorize sulfur-containing substances based on their origin and their use in both preclinical and clinical research, as well as in dietary supplements.

Solving the crystal structure of human calcium-free S100Z: the siege and conquer of one of the last S100 family strongholds.

The X-ray structure of human apo-S100Z has been solved and compared with that of the zebrafish calcium-bound S100Z, which is the closest in sequence. Human apo-S100A12, which shows only 43% sequence identity to human S100Z, has been used as template model to solve the crystallographic phase problem. Although a significant buried surface area between the two physiological dimers is present in the asymmetric unit of human apo-S100Z, the protein does not form the superhelical arrangement in the crystal as observed for the zebrafish calcium-bound S100Z and human calcium-bound S100A4. These findings further demonstrate that calcium plays a fundamental role in triggering quaternary structure formation in several S100s. Solving the X-ray structure of human apo-S100Z by standard molecular replacement procedures turned out to be a challenge and required trying different models and different software tools among which only one was successful. The model that allowed structure solution was that with one of the lowest sequence identity with the target protein among the S100 family in the apo state. Based on the previously solved zebrafish holo-S100Z, a putative human holo-S100Z structure has been then calculated through homology modeling; the differences between the experimental human apo and calculated holo structure have been compared to those existing for other members of the family.

Mitochondrial potassium channels as pharmacological target for cardioprotective drugs.

Brief periods of ischemia are known to confer to the myocardium an increased resistance to the injury due to a later and more prolonged ischemic episode. This phenomenon, known as ischemic preconditioning (IPreC), is ensured by different biological mechanisms. Although an exhaustive comprehension of them has not been reached yet, it is widely accepted that mitochondria are pivotally involved in controlling cell life and death, and thus in IPreC. Among the several signaling pathways involved, as triggers and/or end effectors, in the mitochondrial mechanisms of cardioprotection, an important role is played by the activation of potassium channels located in the mitochondrial inner membrane (mitoK) of cardiomyocytes. Presently, different types of mitoK channels have been recognized in the heart, such as ATP-sensitive (mitoKATP) and calcium-activated (mitoBK(Ca) and mitoSK(Ca)) potassium channels. Consistently, drugs modulating mitoK, on one hand, have been employed as useful experimental tools for early basic studies on IPreC. On the other hand, activators of mitoK are promising and innovative therapeutic agents for limiting the myocardial injury due to ischemic episodes. In this review, we report the experimental evidence supporting the role of mitoK in signaling pathways in the mechanisms of cardioprotection and an overview on the most important molecules acting as modulators of these channels, with their profiles of selectivity. Some innovative pharmaceutical strategies for mitochondriotropic drugs have been also reported. Finally, an appendix describing the main experimental approaches usually employed to study mitoK in isolated mitochondria or in intact cells has been added.

Different patterns of H2S/NO activity and cross-talk in the control of the coronary vascular bed under normotensive or hypertensive conditions.

Hydrogen sulfide (H2S) and nitric oxide (NO) play pivotal roles in the cardiovascular system. Conflicting results have been reported about their cross-talk. This study investigated their interplays in coronary bed of normotensive (NTRs) and spontaneously hypertensive rats (SHRs). The effects of H2S- (NaHS) and NO-donors (sodium nitroprusside, SNP) on coronary flow (CF) were measured in Langendorff-perfused hearts of NTRs and SHRs, in the absence or in the presence of propargylglycine (PAG, inhibitor of H2S biosynthesis), L-NAME (inhibitor of NO biosynthesis), ODQ (inhibitor of guanylate cyclase), L-Cysteine (substrate for H2S biosynthesis) or L-Arginine (substrate for NO biosynthesis). In NTRs, NaHS and SNP increased CF; their effects were particularly evident in Angiotensin II (AngII)-contracted coronary arteries. The dilatory effects of NaHS were abolished by L-NAME and ODQ; conversely, PAG abolished the effects of SNP. In SHRs, high levels of myocardial ROS production were observed. NaHS and SNP did not reduce the oxidative stress, but produced clear increases of the basal CF. In contrast, in AngII-contracted coronary arteries of SHRs, significant hyporeactivity to NaHS and SNP was observed. In SHRs, the vasodilatory effects of NaHS were only modestly affected by L-NAME and ODQ; PAG poorly influenced the effects of SNP. Then, in NTRs, the vascular actions of H2S required NO and vice versa. By contrast, in SHRs, the H2S-induced actions scarcely depend on NO release; as well, the NO effects are largely H2S-independent. These results represent the first step for understanding pathophysiological mechanisms of NO/H2S interplays under both normotensive and hypertensive conditions.

Vasorelaxation by hydrogen sulphide involves activation of Kv7 potassium channels.

Hydrogen sulphide (H2S) has been recently hypothesized to be an endogenous adipocyte-derived relaxing factor, evoking vasorelaxation of conductance and resistance vessels. Although the activation of ATP-sensitive potassium channels is known to play a central role in H2S-induced vasorelaxation, activation of vascular Kv7 voltage-gated potassium channels has also been suggested. To investigate this possibility, the ability of selective activators and blockers of distinct classes of potassium channels to affect vasodilation induced by the H2S-donor NaHS, as well as NaHS-induced Rb(+) efflux in endothelium-denuded rat aortic rings, was investigated. NaHS-induced changes of membrane potential were fluorimetrically assessed on human vascular smooth muscle (VSM) cells. Modulation of Kv7.4 channels by NaHS was assessed by electrophysiological studies, upon their heterologous expression in CHO cells. In isolated aortic rings, NaHS evoked vasorelaxing responses associated with an increase of Rb(+)-efflux. NaHS promoted membrane hyperpolarization of human VSM cells. These effects were antagonized by selective blockers of Kv7 channels. The H2S-donor caused a left-shift of current activation threshold of Kv7.4 channels expressed in CHO cells. Altogether, these results suggest that the activation of Kv7.4 channels is a key mechanism in the vascular effects of H2S. Given the relevant roles played by Kv7.4 channels in VSM contractility and by H2S in circulatory homeostasis regulation, these findings provide interesting insights to improve our understanding of H2S pathophysiology and to focus on Kv7.4 channels as novel targets for therapeutic approaches via the "H2S-system".

The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+ß1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+ß1-subunits. Co-expression of the ß1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+ß1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+ß1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+ß1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the ß1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.

Persulfidation of mitoKv7.4 channels contributes to the cardioprotective effects of the H2S-donor Erucin against ischemia/reperfusion injury.

BACKGROUND: Hydrogen sulfide (H2S) is a gasotransmitter deeply involved in cardiovascular homeostasis and implicated in the myocardial protection against ischemia/reperfusion. The post-translational persulfidation of cysteine residues has been identified as the mechanism through which H2S regulates a plethora of biological targets. Erucin (ERU) is an isothiocyanate produced upon hydrolysis of the glucosinolate glucoerucin, presents in edible plants of Brassicaceae family, such as Eruca sativa Mill., and it has emerged as a slow and long-lasting H2S-donor. AIM: In this study the cardioprotective profile of ERU has been investigated and the action mechanism explored, focusing on the possible role of the recently identified mitochondrial Kv7.4 (mitoKv7.4) potassium channels. RESULTS: Interestingly, ERU showed to release H2S and concentration-dependently protected H9c2 cells against H2O2-induced oxidative damage. Moreover, in in vivo model of myocardial infarct ERU showed protective effects, reducing the extension of ischemic area, the levels of troponin I and increasing the amount of total AnxA1, as well as co-related inflammatory outcomes. Conversely, the pre-treatment with XE991, a blocker of Kv7.4 channels, abolished them. In isolated cardiac mitochondria ERU exhibited the typical profile of a mitochondrial potassium channels opener, in particular, this isothiocyanate produced a mild depolarization of mitochondrial membrane potential, a reduction of calcium accumulation into the matrix and finally a flow of potassium ions. Finally, mitoKv7.4 channels were persulfidated in ERU-treated mitochondria. CONCLUSIONS: ERU modulates the cardiac mitoKv7.4 channels and this mechanism may be relevant for cardioprotective effects.

NO-releasing hybrids of cardiovascular drugs.

Nitric oxide (NO) is an endogenous compound, which plays a fundamental role in the modulation of the function of the cardiovascular system, where it induces vasorelaxing and antiplatelet responses, mainly through the stimulation of guanylate cyclase and the increase of cGMP. Many drugs of common, time-honoured clinical use (for example, glycerol trinitrate and all the vasodilator nitrites and nitrates) act via the release of exogenous NO, thus mimicking the effects of the endogenous factor. In the last few years, a revision of the "one-compound-one-target" paradigm has led pharmacologists and pharmaceutical chemists to develop new classes of molecules which combine different pharmacodynamic properties. This innovative pharmacological/pharmaceutical strategy has produced hybrid drugs, with a dual mechanism of action: a) the slow release of nitric oxide and b) another fundamental pharmacodynamic profile. These drugs have been obtained by inserting appropriate NO-donor chemical groups (i.e. nitrate esters, nitrosothiols, etc.), linked to a known drug, by means of a variable spacer moiety. These new pharmacodynamic hybrids present the advantage of combining a basic mechanism of action (for example, cyclooxygenase inhibition, beta-antagonism or ACE inhibition) with a slow release of NO, which may be useful either to reduce adverse side effects (for example, the gastrotoxicity of NSAIDs), or to improve the effectiveness of the drug (for example, conferring direct vasorelaxing and antiplatelet effects on an ACE-inhibitor). The aim of this review is to present the chemical features of NO-releasing hybrids of cardiovascular drugs, and to explain the pharmacological improvements obtained by the addition of the NO-donor properties.

(+/-)-Naringenin as large conductance Ca(2+)-activated K+ (BKCa) channel opener in vascular smooth muscle cells.

UNLABELLED: BACKGROUND AND PURPOSE. The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/-)-naringenin. EXPERIMENTAL APPROACH: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. KEY RESULTS: (+/-)-Naringenin induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline (pIC(50) values of 4.74 and 4.68, respectively). Tetraethylammonium, iberiotoxin, 4-aminopyridine and 60 mM KCl antagonised (+/-)-naringenin-induced vasorelaxation, while glibenclamide did not produce any significant antagonism. Naringin [(+/-)-naringenin 7-beta-neohesperidoside] caused a concentration-dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (+/-)-naringenin. In rat tail artery myocytes, (+/-)-naringenin increased large conductance Ca(2+)-activated K(+) (BK(Ca)) currents in a concentration-dependent manner; this stimulation was iberiotoxin-sensitive and fully reversible upon drug wash-out. (+/-)-Naringenin accelerated the activation kinetics of BK(Ca) current, shifted, by 22 mV, the voltage dependence of the activation curve to more negative potentials, and decreased the slope of activation. (+/-)-Naringenin-induced stimulation of BK(Ca) current was insensitive either to changes in the intracellular Ca(2+) concentration or to the presence, in the pipette solution, of the fast Ca(2+) chelator BAPTA. However, such stimulation was diminished when the K(+) gradient across the membrane was reduced. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effect of the naturally-occurring flavonoid (+/-)-naringenin on endothelium-denuded vessels was due to the activation of BK(Ca) channels in myocytes.

Structure of chicken plasma retinol-binding protein.

The crystal structure of the specific carrier of retinol (retinol-binding protein, RBP) purified from chicken plasma has been determined (space group P2(1)2(1)2(1), with a=46.06(5) A, b=53.56(6) A, c=73.41(8) A, and one protein molecule in the asymmetric unit). Despite being obtained from a species phylogenetically distant from mammals, chicken holoRBP has an overall structure that closely resembles the previously determined structures of mammalian holoRBPs. The lack in chicken RBP of eight carboxy-terminal amino acid residues characteristic of mammalian RBPs does not significantly affect the protein structure. A distinctive feature of the avian protein is a better definition of the loop 63-67, close to the opening of the beta-barrel cavity accommodating the retinol molecule, which is rather disordered in the structures of mammalian RBPs.

Crystal structure of the catalytic domain of human matrix metalloproteinase 10.

The catalytic domain of matrix metalloproteinase-10 (MMP-10) has been expressed in Escherichia coli and its crystal structure solved at 2.1 A resolution. The availability of this structure allowed us to critically examine the small differences existing between the catalytic domains of MMP-3 and MMP-10, which show the highest sequence identity among all MMPs. Furthermore, the binding mode of N-isobutyl-N-[4-methoxyphenylsulfonyl]glycyl hydroxamic acid (NNGH), which is one of the most known commercial inhibitors of MMPs, is described for the first time.

Amino acid sequence and crystal structure of buffalo alpha-lactalbumin.

Isolation, purification, amino acid sequence determination and X-ray crystal structure of buffalo alpha-lactalbumin were performed in order to gain further knowledge of the molecular basis of alpha-lactalbumin in the lactose synthase complex. The deduced amino acid sequence differs at one position from the bovine alpha-lactalbumin sequence (at position 17). The refined crystal structure at 2.3 A is very similar to those previously reported for human and baboon alpha-lactalbumins. However, a portion of the molecule (residues 105-109) exhibits different conformation. It forms a 'flexible loop', and appears to be a functionally important region in forming the lactose synthase complex.

Large-conductance, ca(2+)-activated k(+) channels: function, pharmacology and drugs.

Because of the physiological role played by the hyperpolarisation process resulting from a K(+) outflow, it is not surprising that compounds able to activate outward K(+) channels are considered as promising drugs, with exciting perspectives for the treatment of several cardiovascular, respiratory, neurological and urological diseases. Among the different and numerous K(+) channel families, medicinal chemistry has focused its major interest onto two channel types: the ATP-sensitive channels (K(ATP)) and the large conductance subtype (BK), that belongs to the wide family of calcium-activated K(+) channels. BK channels are almost ubiquitous and exhibit single channel conductance of 100-300 pS, a property which justifies the potent role of these channels in the control of the membrane potential. BK channels have been investigated as potential therapeutic targets for different neuropathies, because of their profound influence on the neuronal activity. Moreover, BK channels are expected to have applications for the therapy of cardiovascular diseases. A potent feed-back control of the vascular and non-vascular smooth muscle tone is mediated by these channels, whose activation can be promoted by both a rise of the intracellular free calcium concentration as well as a membrane depolarisation. Additionally, BK channel activation can also be induced by other factors, such as cAMP-mediated phosphorylation, G-proteins, GMP and cGMP. The aim of this paper is to give a concise overview of the biological and pharmacological properties and potential therapeutic applications of activators of BK channels present at the vascular level. The "state of the art" in the pharmaceutical development of natural and synthetic BK-activators, with a description of the lead chemical structures, will be also described.

Torsadogenic cardiotoxicity of antipsychotic drugs: a structural feature, potentially involved in the interaction with cardiac HERG potassium channels.

Many non-cardiovascular drugs of common clinical use cause, as an unwanted accessory property, the prolongation of the cardiac repolarisation process, due to the block of the HERG (Human Ether-a-go-go Related Gene) potassium channel, responsible for the repolarising I(Kr) current. This delayed cardiac repolarisation process can be often unmasked by a prolongation of the QT interval of the ECG. In these conditions, premature action potentials can generate morphologically anomalous after-polarisations, and trigger a dangerous kind of polymorphic ventricular tachyarrhythmia, known as torsade de pointes, which can evolve in ventricular fibrillation and death. The risk associated with the torsadogenic cardiotoxicity of drugs, which prolong the QT interval has been the topic of documents produced by many health authorities, giving important issues about the preclinical and clinical evaluation of cardiac safety. Besides, public and private research laboratories developed several experimental in vitro or in vivo strategies, aimed to an early recognition of the influence of a drug (or of a drug-candidate) on the HERG channel and/or on the cardiac repolarisation process. Also the identification of a possible pharmacophore model, common in all or at least in numerous torsadogenic drugs, could represent a first step for the development of useful in silico approaches, allowing a preliminary indication about the potential torsadogenic property of a given molecule. In this work, we described the electrophysiological basis of torsade de pointes and listed several pharmacological classes of torsadogenic drugs. Among them, we focused our attention on antipsychotics, with an accurate overview on the experimental and clinical reports about their torsadogenic properties. Moreover, a common structural feature exhibited by these drugs, despite of their remarkable chemical differences, is evidenced by a computational approach and is indicated as a possible "facilitating" requirement for their torsadogenic properties. Together with other remarks, coming from different computational studies, the individuation of a satisfactory "toxicophore" model could be greatly useful, for the theoretical prediction of torsadogenic properties of a given chemical moiety and for the design of new drugs devoid of such an undesired and potentially lethal side-effect.

Adenosine-mediated hypotension in in vivo guinea-pig: receptors involved and role of NO.

1. Adenosine produced a biphasic lowering of the mean BP with a drastic bradycardic effect at the highest doses. The first phase hypotensive response was significantly reduced by the nitric oxide (NO) synthase inhibitor L-NAME. 2. The A(2a)/A(2b) agonist NECA produced hypotensive and bradycardic responses similar to those elicited by adenosine, which were not significantly modified by the A(2b) antagonist enprofylline. 3. The A(2a) agonist CGS 21680 did not significantly influence basal HR while induced a hypotensive response antagonized by the A(2a) selective antagonist ZM 241385, and reduced by both L-NAME and the guanylate cyclase inhibitor methylene blue. 4. The A(1) agonist R-PIA showed a dose-dependent decrease in BP with a drastic decrease in HR at the highest doses. The A(1) selective antagonist DPCPX significantly reduced the bradycardic activity and also the hypotensive responses obtained with the lowest doses while it increased those obtained with the highest ones. 5. The A(1)/A(3) agonist APNEA, in the presence of the xanthinic non-selective antagonist 8-pSPT, maintained a significant hypotensive, but not bradycardic, activity, not abolished by the histamine antagonist diphenhydramine. 6. The selective A(3) agonist IB-MECA revealed a weak hypotensive and bradycardic effect, but only at the highest doses. 7. In conclusion, in the systemic cardiovascular response to adenosine two major components may be relevant: an A(2a)- and NO-mediated hypotension, and a bradycardic effect with a consequent hypotension, via atypical A(1) receptors. Finally, an 8-pSPT-resistant hypotensive response not attributable to A(3) receptor-stimulation or to release of histamine by mastocytes or other immune cells was observed.

Influence of depolarization on vasorelaxant potency and efficacy of Ca2+ entry blockers, K+ channel openers, nitrate derivatives, salbutamol and papaverine in rat aortic rings.

We examined the effects of various KCl concentrations on the actions of some vasodilators belonging to different pharmacological classes in rat aortic rings. In some experiments, tissues were precontracted with noradrenaline after blocking voltage-dependent channels to assess the effects of depolarisation unaccompanied by the entry of extracellular Ca2+ into the cytosol. Concentration/response curves for the vasorelaxant effect of calcium entry blockers (e.g. diltiazem), K+ channel openers (e.g. aprikalim), nitrate derivatives (e.g. nitroglycerin), a beta2-adrenergic agonist (salbutamol) and papaverine were obtained by using endothelium-denuded rat aortic rings precontracted with KC1 (20-60 mM) to determine the potencies and efficacies of the drugs. The efficacies and potencies of calcium entry inhibitors were virtually independent of the [KCl]. A reduction in the potency (up to 18-fold) of papaverine occurred without changes in efficacy when the [KCl] was raised from 20 to 60 mM. The decline in potency was even greater for nitrate-like compounds. The potency of K+ channel openers in aortic rings precontracted with 30 mM KCI decreased by three- to sixfold compared with those precontracted with 20 mM KCl. With the exception of pinacidil, the efficacy of these agents already started to decline in preparations precontracted with 25 mM KCI and was virtually zero in preparations precontracted with 60 mM KCI. In contrast to other K+ channel openers, the vasorelaxant action of pinacidil was relatively resistant to glibenclamide. Salbutamol produced only a slight relaxation even in preparations precontracted with 20 mM KCl. In nitrendipine-pretreated, noradrenaline-precontracted aortic rings, the vasorelaxant effects of aprikalim, but not those of linsidomine or papaverine, declined when the [KCl] of the bathing medium was increased. In conclusion, the vasorelaxant potency and efficacy of calcium entry blockers is independent of the [KCI] used to precontract rat aortic rings, and thus, of the degree of membrane depolarisation. In contrast, increasing the [KCl] strongly reduces the potency and the efficacy of K+ channel openers not only in this preparation but also in noradrenaline-precontracted rings in which the entry of extracellular Ca2+ was prevented with nitrendipine. This indicates that, with the exception of pinacidil, the vasorelaxant activity of K+ channel openers depends on the degree of membrane depolarisation. Finally, the vasorelaxant potency and efficacy of nitrate-like compounds and papaverine are independent of depolarisation per se but they are markedly affected by the influx of Ca2+ accompanying elevated [KCI]. Thus, the degree of vessel depolarisation should be taken into consideration when attempting to compare potencies and efficacies among vasorelaxant agents.

Experimental and theoretical comparisons between the classical Schild analysis and a new alternative method to evaluate the pA2 of competitive antagonists.

The Schild analysis is undoubtedly the most frequently used powerful diagnostic tool to investigate the nature of an antagonist and, consequently, to evaluate its potency, often expressed as pA2. Nevertheless, different reasons often prevent the experimenter from applying this analysis, leading to use an inhibition curve for the antagonist and to evaluate its potency by means of several approaches, which are generally considered theoretically invalid. In a recent work, a new theoretical approach, mathematically analogous to the Schild one, has been shown. By means of a simplified experimental protocol based on an antagonist inhibition curve (following a control concentration-response curve for the agonist), this method allows a linear regression analysis, giving a slope value absolutely equivalent to the Schild slope and a reliable estimation of the pA2 of a competitive antagonist. In this paper, this new method has been compared with the Schild analysis, to determine the parameters of potency relative to well-known competitive antagonists, on different in vitro isolated preparations. In strips of guinea pig isolated gastric smooth muscle, pirenzepine antagonised the effects of bethanechol. In guinea pig isolated ileum, atropine blocked the contracturant effects of carbamylcholine, while in electrostimulated ileum segments, the inhibitory responses to alpha-methylnoradrenaline were reduced by idazoxan. Finally, in guinea pig isolated spontaneously beating atria, the negative inotropic effects of 5'-N-ethylcarboxamidoadenosine were antagonised by 8-cyclopentyl-1,3-dipropylxanthine. The parameters of potency, relative to all the above competitive antagonists and expressed as pA2, resulted almost equivalent, when calculated by the Schild analysis or by the alternative method. Furthermore, when tested also for the well-known irreversible alkylating agent dibenamine in rat aortic rings stimulated by noradrenaline, the alternative method furnished a profile of clear nonlinearity, unmasking the nature of the antagonism. Finally, the relationships between the results calculated by the alternative analysis or by the Schild analysis and different levels of computer-generated "random noise" (affecting the shape and the position of theoretical curves) were also evaluated, in order to know the robustness of the new method. The two methods proved reliable and almost equivalent in robustness, when applied with different levels of "random noise". These results confirm the Schild analysis as the most accurate tool to study antagonists, since this analysis can furnish the highest number of information and observations on the behaviour of an antagonist. Nevertheless, when limiting conditions prevent a classical Schild analysis and impose the use of an inhibition curve, the new method probably represents the most preferable experimental approach. Indeed, it allows to calculate the antagonist potency, after the evaluation of a slope parameter giving an important information about the possible nature of the antagonism.

An alternative method to evaluate the nature of an antagonist and its potency: a theoretical approach.

The Schild analysis is certainly the most reliable method for antagonism studies. The Schild regression allows one to determine the parameter of the Schild-slope, which represents a powerful diagnostic tool when investigating the nature of an antagonist and, consequently, to evaluate its potency. Nevertheless, in functional pharmacology, often practical reasons lead the experimenter to obtain an inhibition curve for the antagonist and calculate its potency by means of equations, which can be considered as a derivation of the Cheng-Prusoff analysis. This approach is considered theoretically invalid, because it does not allow to know the exact nature of the antagonism, and thus the evaluation of the antagonist dissociation constant can be meaningless. In this paper, a new method is proposed, which, by means of an equation closely similar to the Schild one, permits one to obtain a linear regression analysis, giving a slope value absolutely equivalent to the Schild-slope. This method allows us to determine both the nature and the potency of an antagonist, and requires an experimental procedure substantially analogous to the one performed to obtain an inhibition curve.