On contemporaneous controls, unlikely outcomes, boxes and replacing the 'Student': good statistical practice in pharmacology, problem 3.

This paper is intended to assist pharmacologists to make the most of statistical analysis and avoid common errors. A scenario, in which an experimenter performed an experiment in two separate stages, combined the control groups for analysis and found some surprising results, is presented. The consequences of combined controls are discussed, appropriate display and analysis of the data are described, and an analysis of the likelihood of erroneous conclusions is made. Comparisons between data from separately conducted experimental series are hazardous when there is any possibility that the properties of the experimental units have changed between the series. Experiments that have been performed independently should be analyzed independently. Unlikely or surprising results should be treated with caution and a high standard of evidence should be required, and verification by repeated experiments should be performed and reported. Box and whisker plots contain more information than plots more commonly used to display for qualitative variables and should be used where the sample size is large enough (say, n > or = 5). In most biomedical experiments the observations are not random samples from large populations as assumed by conventional parametric analyses such as Student's t-test, and so permutation tests, which do not lose their validity when a sampled population is non-normal or when the data are not random samples, should frequently be used instead of Student's t-tests.

Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens.

1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

Dynamic mechanisms of non-classical antagonism by competitive AT(1) receptor antagonists.

Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.

Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.

The assessment of antagonist potency under conditions of transient response kinetics.

The muscarinic acetylcholine receptor antagonists, atropine and pirenzepine, produced an apparent insurmountable antagonism of muscarinic M(1) receptor-mediated intracellular Ca(2+) mobilization in Chinese hamster ovary (CHO) cells when tested against the agonists carbachol or xanomeline. Each antagonist caused a dextral shift of the agonist concentration-response curves with depression of the maximum response that was incomplete (i.e., saturated) and which varied with the pairs of agonist and antagonist. Equilibrium competition binding assays found no deviation from simple, reversible competitive behavior for either antagonist. The relative rates of dissociation of unlabeled atropine and pirenzepine were also assessed in radioligand kinetic studies and it was found that atropine dissociated from the receptor approximately 8-fold slower than pirenzepine. Numerical dynamic simulations suggested that the insurmountability of antagonism observed in the present study was probably a kinetic artifact related to the measurement of transient responses to a non-equilibrated agonist in the presence of a slowly dissociating antagonist. Importantly, the patterns of antagonism observed included a saturable depression of agonist maximal response, a mode of antagonism that is incompatible with the previously described phenomenon of hemi-equilibrium states. Monte Carlo simulations indicated that reasonable, semi-quantitative estimates of antagonist potency could be determined by a minor modification of standard methods, where equieffective agonist concentrations, rather than EC(50) values, are compared in the absence and presence of antagonist. Application of the latter approach to the functional data yielded estimates of antagonist potency that were in excellent agreement with those derived from the equilibrium binding assays, thus indicating that the present method can be useful for quantifying antagonist potency under non-equilibrium conditions.

Examination of adenosine receptor-mediated relaxation of the pig coronary artery.

1. The adenosine receptors mediating relaxation of porcine isolated left anterior descending coronary arteries (LAD) and the effects of the level and type of preconstriction on the responses to adenosine analogues were examined in the present study. 2. Relaxation responses to the non-selective adenosine receptor agonist N-ethylcarboxamidoadenosine (NECA) were endothelium independent. N-Ethylcarboxamidoadenosine, GR 79236 (A1 receptor selective) and 8-cyclopentyl-1,3-dipropylxanthine (CGS 21680) (A2A receptor selective) produced full relaxation in LAD precontracted to 50% of the response to potassium depolarization with the thromboxane receptor agonist U46619. The order of potency was CGS 21680 = NECA > GR 79236, consistent with that defining the A2A receptor subtype. 3. 3,7-Dimethyl-1-propargylxanthine (DMPX; A2 receptor selective) competitively antagonized NECA and CGS 21680 with pKB values of 4.95 +/- 0.09 and 5.06 +/- 0.22, respectively. The A1 receptor selective antagonist 1,3-[3H]-dipropyl-8-cyclopentylxanthine (DPCPX) had no effect on NECA relaxation, even in the presence of DMPX. 4. The sensitivity to relaxation by NECA was dependent on the precontracting agent. Arteries precontracted with endothelin (ET)-1 were most sensitive to NECA, U46619-precontracted arteries were intermediate and KCl-precontracted arteries were least sensitive. 5. The potency of NECA was reduced when the preconstriction level was increased from 50 to 90% of maximum in U46619-precontracted arteries (pEC50 7.94 +/- 0.12 and 7.35 +/- 0.04, respectively) and, in KCl-precontracted arteries, both the potency and maximum effect of NECA were reduced when the preconstriction level increased from 50 to 80% of maximum (pEC50 7.52 +/- 0.13 and 6.91 +/- 0.26, respectively; maximum responses 82.5 +/- 10.2 and 23.9 +/- 3.6%, respectively, of the preconstricted tone). Relaxation responses to NECA were independent of the level of precontraction in ET-1-precontracted arteries. 6. In porcine LAD, relaxation responses to adenosine analogues were endothelium independent and were mediated via A2A adenosine receptors. Responses to NECA were dependent on both the level and type of preconstriction.

Roles of key functional groups in omega-conotoxin GVIA synthesis, structure and functional assay of selected peptide analogues.

The contributions of various functional groups to the pharmacophore of the N-type calcium-channel blocker, omega-conotoxin GVIA (GVIA), have been investigated using structural and in-vitro functional studies of analogues substituted at one or two positions with non-native residues. In most cases the structure of the analogue was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) were employed to monitor N-type calcium-channel activity. The data provide a more detailed picture of the roles in GVIA structure and activity of the crucial Lys2 and Tyr13, as well as all other positively charged residues, Tyr22, the hydroxyproline residues and the C-terminal amido moiety, many of which were identified as being important for activity in an alanine scan [Lew et al. (1997) J. Biol. Chem. 272, 12014-12023]. Substitutions of Lys2 with nonstandard amino acids and arginine quantified the roles of the length and charge of the Lys side chain. The orientation of the Tyr13 side chain and its hydroxyl moiety was shown to be important by substitution with d-Tyr and the d-form and l-form of the constrained analogue 7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [Tic(OH)]. The roles of the Hyp10 and Hyp21 hydroxyl groups, investigated by proline substitutions, appear to be more structural (as monitored by NMR) than functional, although small decreases in potency were observed in some assays. The reversibility of the channel blockade was also studied, and several analogues with faster wash-out characteristics than native GVIA were identified. Rapid reversibility (as in the case of omega-conotoxin MVIIA) may be beneficial for therapeutic applications. Disubstituted analogues revealed some interesting cooperative effects, which were not predicted from single-residue substitutions. A disubstituted chimera of GVIA and omega-conotoxin MVIIA was more potent than either native molecule. The more detailed description of the GVIA pharmacophore obtained here provides a better basis for the future design of truncated peptide and peptidomimetic analogues.

Mechanisms of melatonin-induced vasoconstriction in the rat tail artery: a paradigm of weak vasoconstriction.

1. Vasoconstrictor effects of melatonin were examined in isolated rat tail arteries mounted either in an isometric myograph or as cannulated pressurized segments. Melatonin failed by itself to mediate observable responses but preactivation of the arteries with vasopressin (AVP) reliably uncovered vasoconstriction responses to melatonin with maxima about 50% of maximum contraction. Further experiments were conducted with AVP preactivation to 5-10% of the maximum contraction. 2. Responses to melatonin consisted of steady contractions with superimposed oscillations which were large and irregular in isometric but small in isobaric preparations. Nifedipine (0.3 microM) reduced the responses and abolished the oscillations. Charybdotoxin (30 nM) increased the magnitude of the oscillations with no change in the maximum response. 3. Forskolin (0.6 microM) pretreatment increased the responses to melatonin compared to control and sodium nitroprusside (1 microM) treated tissues. The AVP concentration required for preactivation was 10 fold higher than control in both the forskolin and nitroprusside treated groups. 4. In isometrically-mounted arteries treated with nifedipine, melatonin receptor agonists had the potency order 2-iodomelatonin > melatonin > S20098 > GR196429, and the MT2-selective antagonist luzindole antagonized the effects of melatonin with a low pK(B) of 6.1+/-0.1. 5. It is concluded that melatonin elicits contraction of the rat tail artery via an mt1 or mt1-like receptor that couples via inhibition of adenylate cyclase and opening of L-type calcium channels. Calcium channels and charybdotoxin-sensitive K channels may be recruited into the responses via myogenic activation rather than being coupled directly to the melatonin receptors. 6. It is proposed that the requirement of preactivation for overt vasoconstrictor responses to melatonin results from the low effector reserve of the melatonin receptors together with the tail artery having threshold inertia. Potentiative interactions between melatonin and other vasoconstrictor stimuli probably also result from the threshold inertia. A simple model is presented and a general framework for consideration of interactions between weak vasoconstrictor agonists and other vasoconstrictor stimuli is discussed.

Structure-function relationships of omega-conotoxin GVIA. Synthesis, structure, calcium channel binding, and functional assay of alanine-substituted analogues.

The structure-function relationships of the N-type calcium channel blocker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

Synthesis and characterization of a selective peptide antagonist of neuropeptide Y vascular postsynaptic receptors.

1. A cyclic dimeric nonapeptide neuropeptide Y (NPY) receptor antagonist, 1229U91, was synthesized by Fmoc chemistry and dimerised in solution. Its effects were assayed in mesenteric arteries from rats and mice, and in rat vas deferens. 2. Mesenteric arteries were cannulated and pressurised to 55 mmHg and the external diameters continuously measured. NPY, PYY, Leu31Pro34NPY and NPY(13-36) each caused concentration-related contractions with the order of potency PYY > or = Leu31Pro34NPY = NPY > NPY (13-36), consistent with the Y1 receptor subtype. 3. 1229U91 had no agonist activity in the arteries but caused a concentration-related rightward shift of NPY (mouse arteries) or Leu31Pro34NPY (rat) concentration-response curves. The antagonism was competitive with pKBS of 7.69 +/- 0.15 and 7.47 +/- 0.13 in the mouse and rat arteries, respectively. 4. Sympathetic nerves in the vas deferens were stimulated with a single electrical field pulse every 20 s and the twitch responses recorded. NPY, PYY, Leu31Pro34NPY and NPY(13-36) inhibited the twitches with the order of potency PYY > NPY > NPY(13-36) >> Leu31Pro34NPY, consistent with the Y2 receptor subtype. 5. 1229U91 inhibited the vas deferens twitch with a shallow concentration-response curve and a time-course of inhibition distinct from that of NPY. 1229U91 (30 microM) did not cause a rightward shift of the NPY concentration-response curve. 1229U91 is at least 5 orders of magnitude less potent in the vas deferens than in rat brain Y2 binding assays reported by others, suggesting that the brain and vas deferens Y2 receptors are different. 6. It is concluded that 1229U91 is a competitive antagonist of NPY Y1 vascular receptors and has additional properties that inhibit the electrically evoked twitch of the rat vas deferens.

Effect of cold storage in University of Wisconsin solution on the responses of porcine hepatic arteries to 5-hydroxytryptamine and bradykinin in vitro.

1. Responses to 5-hydroxytryptamine (5-HT), bradykinin and sodium nitroprusside (SNP) were examined in hepatic arteries of the pig 1 h after dissection (fresh) and following 24 h storage in either Ca(2+)-free Krebs solution or the cryopreservative University of Wisconsin (UW) solution. 2. In fresh arteries contracted to approximately 40% of the maximum response to potassium with U46619, a thromboxane A2-mimetic, concentration-response curves to 5-HT (10(-10)-10(-5) M) were biphasic, with relaxation at low concentrations (< 10(-8) M) and contraction at high concentrations. Bradykinin (10(-10)-10(-7) M) produced concentration-dependent relaxation of precontracted fresh arteries with no apparent constrictor response. 3. Following 24 h storage in Ca(2+)-free Krebs solution, relaxation responses to 5-HT and the sensitivity of the arteries to bradykinin were significantly reduced. Storage in UW solution did not affect relaxation responses to either 5-HT or bradykinin. Relaxation responses to SNP (10(-8)-10(-3) M) were unaffected by storage in either solution. 4. Treatment of fresh arteries with NG-nitro-L-arginine (L-NOARG, 10(-4) M) significantly attenuated the relaxation response to 5-HT and displaced the bradykinin concentration-response curve four fold to the right with no affect on its maximum relaxation. 5. From these results it is concluded that endothelial cell function is better preserved during cold storage in UW solution than in Ca(2+)-free Krebs solution.

Synthesis and biological characterization of a series of analogues of omega-conotoxin GVIA.

The 27-residue polypeptide omega-conotoxin GVIA (omega-CgTx), from the venom of the cone shell Conus geographus, blocks N-type neuronal calcium channels. It contains three disulphide bridges. We report here the synthesis and biological characterization of a series of analogues in which one disulphide has been replaced by substitution of appropriate Cys residues with Ser, viz. [Ser1,16]-omega -CgTx, [Ser8,19]-omega-CgTx, [Ser15,26)-omega-CgTx, [Ser16]-omega-CgTx8-27 and [Ser15]-omega-CgTx1-19. All syntheses were conducted manually using either Boc or Fmoc methodology. Deprotected peptides were oxidized to their bridged forms using either aerial oxidation or aqueous dimethyl sulphoxide. Peptides were purified using RP-HPLC, and their purity and identity were checked by RP-HPLC, capillary electrophoresis and mass spectrometry. Inhibition of neuronal N-type calcium channels was assessed as the inhibition of the twitch responses of rat vas deferens stimulated with single electrical pulses at 20 second intervals. None of these analogues was biologically active, suggesting that the disulphides play an important role in maintaining biological activity.

Analysis of competitive agonist-antagonist interactions by nonlinear regression.

The rigorous estimation of a dissociation constant (Kb) for antagonists in functional assays has been sought by pharmacologists using a variety of techniques ever since the regression method of Arunlakshana and Schild in 1959. Here, Michael Lew and James Angus describe a simplified global regression method with improved accuracy compared to Schild analysis. The method is suitable for personal computers with standard graphing and statistical software. The accuracy of the predicted pKb values and confidence intervals has been tested by comparing examples of published data, and by mathematical (bootstrap) simulations.

Extended concentration-response curves used to reflect full agonist efficacies and receptor occupancy-response coupling ranges.

1. An approach is described for generating extended agonist concentration-response curves where the responses are unconstrained by the normal tissue maximum response. Functional antagonism is employed to hold the tissue state in the range where any change in stimulus can be translated into a measurable response. 2. The maximum response of these extended concentration-response curves provides an index of intrinsic activity reflecting the agonist efficacy and the receptor occupancy-response coupling range. 3. The use of this approach is illustrated with extended concentration-response curves for noradrenaline (NA), vasopressin, acetylcholine (ACh), and 5-methylfurmethide in the small mesenteric and tail arteries of the rat. Both NA and vasopressin can maximally activate the arteries, but the new protocol shows that NA can produce more cellular activation than vasopressin in the tail artery. Both ACh and 5-methylfurmethide are full agonists but ACh has a higher intrinsic activity than 5-methylfurmethide. The ACh muscarinic receptors in the mesenteric artery have a larger occupancy-response range than the ACh muscarinic-receptors in the tail artery, and the alpha-adrenoceptors in the tail artery appear to have a larger occupancy-response coupling range than those in the mesenteric artery. 4. This approach extends our ability to compare the efficacies of full agonists, and to compare the occupancy-response coupling ranges of receptors that can normally maximally activate the assay tissue. This is achieved without the use of an irreversible antagonist and should be applicable to many receptors and pharmacological assay systems where responses are stable and functional antagonists are available.

The effect of high perfusion rates on the endothelial diffusion barrier in rat mesenteric arteries in vitro.

1. Arteries from Wistar-Kyoto rat mesenteries were cannulated with glass cannulae and perfused with Krebs' solution at controlled rates and distending pressure. Arterial diameter was measured and concentration-response curves generated with agonists applied via the perfusate (luminal) and via the solution bathing the outside of the preparation (extraluminal). 2. The alpha 1-adrenoceptor selective agonist methoxamine was about three times more potent when applied extraluminally than intraluminally in arteries perfused at either 25 or 500 microL/min, but the lipophilic alpha 1-adrenoceptor selective agonist SKF 89748-A was equally potent with either mode of application. 3. The extraluminal/intraluminal potency differentials for both methoxamine and noradrenaline were abolished by perfusion for 30 min at a rate calculated to give a high shear stress (100 dyn/cm2). 4. The potency of luminally applied acetylcholine was reduced less than two-fold by the high shear perfusion, and the ability of acetylcholine to maximally relax the arteries was unchanged. 5. It is concluded that (i) the endothelium of small arteries reduces the potency of luminally applied lipophobic agonists by acting as a diffusion barrier; (ii) the ability of the endothelium to act as a diffusion barrier is easily damaged by shear stress in vitro; and (iii) the ability of acetylcholine to relax arteries in vitro is a poor index of the full functioning of the endothelium.

Interpretation of the acetylcholine test of endothelial cell dysfunction in hypertension.

BACKGROUND: In the hypertensive circulation, endothelial cells may release less nitric oxide or more endothelin-1, both powerful vasoactive substances, suggesting an attractive hypothesis for the initiation or reinforcement of hypertension. These substances, however, are not the only way that endothelial dysfunction could be involved in hypertension. In this work we examine the role of the endothelium as a diffusion barrier to vasoconstrictor substances, as a metabolic barrier and as a secretory source of paracrine hormones. REVIEW OF DATA: The evidence that endothelial cell dysfunction occurs in different forms of hypertension comes mainly from the loss of relaxation revealed by the 'acetylcholine test' in a variety of preparations. We examined the strength of this evidence in terms of the stability of the agonist, equilibrium between agonist and receptor, and variations in acetylcholine and other receptor populations on endothelial and smooth muscle cells. The range (Emax) and sensitivity (EC50) of the acetylcholine test was considered in a novel approach to determine the full range by in vitro assay. Using conscious rabbits, we showed that the vascular amplifier of resistance in the hypertensive bed can lead to misinterpretation of changes in reactivity. CONCLUSION: The question of endothelial dysfunction in hypertension as determined by the acetylcholine test is far from proven.

Wall thickness to lumen diameter ratios of arteries from SHR and WKY: comparison of pressurised and wire-mounted preparations.

Passive properties (diameter, wall-to-lumen ratio and axial length) of small mesenteric arteries from SHR and WKY rats were measured with the artery segments cannulated and pressurised, or mounted on wires in a myograph. The measurements were made with a range of distending pressures (or calculated equivalent distending pressures when wire-mounted) from 0 to 180 mm Hg. The axial length of artery segments increased with increasing distending pressure when cannulated, but not when wire-mounted. The axial extension was greater for arteries from WKY (up to 105%) than for arteries from SHR (up to 65%). The arteries from SHR had significantly smaller diameters and greater wall-to-lumen ratios than the arteries from WKY. However, the diameters calculated for the arteries when wire-mounted were less than the measured diameters, and the wall-to-lumen ratio was always greater when wire-mounted than when cannulated because of the underestimated diameter and the absence of axial extension. Wall-to-lumen ratios decreased with increased distending pressure; values at 180 mm Hg were only 18 and 25% of those at 0 mm Hg for WKY and SHR arteries, respectively. The large degree of variability of wall-to-lumen ratios obtained from the two different preparations and the large range of values that are obtained from a single artery at different distending pressures must call into question the validity of characterising vascular hypertrophy by any single estimation of this parameter.