neuropeptide-y and benextramine

Several physiological stimuli cause a rise in intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes. This increased [Ca2+]i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or nonadrenergic neurons, on Ca2+ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with 45CaCl2 for 1 h. Then, the fractional release of 45Ca2+ from the cells was measured. NPY stimulated the efflux of 45Ca2+ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10(-8) M to 10(-6) M). NPY (10(-6) M)-induced Ca2+ efflux was 2.0 +/- 0.16% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by Y1 receptor agonist, [Leu31, Pro34]NPY, but not by Y2 receptor agonist, NPY13-36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY18-36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca2+ efflux from cardiomyocytes is mediated through Y1 receptors. It was also observed that NPY caused a rise in [Ca2+]i to almost 150 nM. NPY-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, NPY caused a 22Na+ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na+/Ca2+ exchange inhibitors. In addition, isoproterenol also caused 45Ca2+ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y1 receptors with which NPY may couple during Na+/Ca2+ exchange.

Topics: Adenosine Triphosphatases; Animals; Calcium; Cells, Cultured; Cystamine; Heart; Male; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Sodium

The putative M1 muscarinic receptor agonist McN-A-343 evoked a dose-dependent increase in mean arterial pressure (MAP) when administered intravenously to conscious freely-moving rats pretreated with the ganglionic nicotinic receptor antagonist pentolinium. A tachycardia accompanied the increase in MAP which was blocked by the beta-adrenergic receptor antagonist propranolol. The increase in MAP was attenuated by the alpha 1-adrenergic receptor antagonist prazosin combined with the alpha 2-adrenergic receptor antagonist yohimbine. Adding propranolol to alpha-adrenergic receptor blockade uncovered a latent pressor response. Replacing prazosin with benextramine (which blocks NPY in addition to alpha-adrenergic receptors) attenuated the pressor response unmasked by propranolol. This attenuation was comparable to that provided by benextramine of the pressor response to intravenous administration of NPY. Adrenal demedullation only slightly attenuated the pressor response while having no effect on the tachycardia. The catecholamine depletor guanethidine greatly attenuated the McN-A-343-evoked increase in MAP and heart rate. The combination of adrenal demedullation and guanethidine did not further attenuate the increase in MAP but did provide better attenuation of the tachycardia than guanethidine alone. These results show that McN-A-343-evokes an increase in MAP and heart rate of conscious freely-moving rats primarily by causing the release of catecholamines, and possibly NPY, from sympathetic neurons with the adrenal glands playing a minor role.

Topics: (4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethylammonium Chloride; Adrenal Medulla; Adrenergic Agonists; Adrenergic Antagonists; Animals; Antihypertensive Agents; Blood Pressure; Cystamine; Drug Interactions; Guanethidine; Male; Muscarinic Agonists; Neuropeptide Y; Prazosin; Propranolol; Rats; Rats, Sprague-Dawley; Yohimbine

1. In order to characterize the neuropeptide Y (NPY) Y1 receptors known to be present in rabbit isolated vas deferens and saphenous vein, the pharmacological activity of the selective NPY Y1 receptor agonists, [Leu31,Pro34] NPY and various other peptide agonists, together with the putative NPY antagonist, benextramine, were compared in the two tissues. 2. In rabbit isolated saphenous vein, cumulative dose-response curves to various NPY agonists were obtained. All the peptides tested caused contractions which developed quite slowly. The rank order of potency obtained was: PYY > NPY > [Leu31,Pro34] NPY = NPY2-36 > hPP >> NPY13-36 = NPY18-36. Incubation with benextramine (BXT) at 100 microM for 30 min irreversibly abolished the contractile response to [Leu31,Pro34] NPY but was ineffective against NPY18-36-induced contractions. 3. Cumulative dose-response curves to [Leu31,Pro34] NPY were performed in the same preparation before and after incubation with 100 microM BXT for 20 min in order to inactivate NPY Y1 receptors. The pKA (-logKA) estimation for [Leu31,Pro34] NPY was 7.60 +/- 0.30 using the operational model and 7.20 +/- 0.33 using the null method; the difference between the two methods was not statistically significant (P = 0.36). 4. Prostatic segments of rabbit vas deferens were electrically stimulated with single pulses. Immediately after stabilization of the contractile response, a cumulative dose-response curve to various NPY agonists was obtained in each tissue. The rank order of potency for twitch inhibition was: PYY> [Leu31,Pro34]NPY > NPY > hPP>NPY2- 36 >>NPY13-36>> NPY 18-36 which indicates the presence of a prejunctional NPY Y1 receptor. BXT at 100 microM incubated for 10 or 60 min did not antagonize the response to[Leu31,Pro34] NPY.5. We conclude that rabbit isolated saphenous vein contains a population of post-junctional NPY Y1 receptors irreversibly blocked by BXT, as well as a population of post-junctional NPY Y2 receptors,which are insensitive to BXT. In contrast, the rabbit isolated vas deferens express a pre-junctional NPYY1 receptor subtype which is not blocked by BXT. Tetramine disulphides such as BXT could be useful tools in classifying NPY receptors.

Topics: Animals; Culture Techniques; Cystamine; Humans; Male; Neuropeptide Y; Peptides; Rabbits; Receptors, Neuropeptide Y; Saphenous Vein; Vas Deferens

1. The pharmacological activity of neuropeptide Y (NPY) and some analogues in inhibiting the twitch contractions induced by electrical stimulation (single pulses at 25 V, 0.15 Hz, 1 ms) in the prostatic portion of the rat isolated vas deferens was investigated. The rank order of agonist potency was: PYY > NPY2-36 > NPY >> NPY13-36 >> NPY18-36 >> [Leu31,Pro34]NPY = hPP, which is consistent with the activation of a Y2 receptor. 2. The putative Y1 and Y2 antagonist, benextramine (BXT), incubated at 100 microM for 10 or 60 min, was ineffective against PYY-induced inhibition of the twitch response, suggesting that the prejunctional Y2 receptor in this tissue is different from the postjunctional one reported in the literature to be sensitive to BXT blockade. 3. The putative NPY antagonist, PYX-2, incubated at 1 microM for 20 min, was completely ineffective in antagonizing PYY-induced inhibition of twitches. 4. The twitch response was totally inhibited by suramin (100 microM) but was little affected by prazosin (1 microM). Furthermore, NPY was without effect on the dose-response curve to ATP in resting conditions. Taken together, these results suggest that in our paradigm, NPY inhibits the release of a purinergic neurotransmitter which mediates contraction of the prostatic portion of the rat vas deferens.

Topics: Adenosine Triphosphate; Adrenergic alpha-Antagonists; Animals; Cystamine; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Neuropeptide Y; Peptide Fragments; Prostate; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Suramin; Vas Deferens

The role of noradrenergic alpha-1 receptors in increasing the behavioural activity after neuropeptide Y (NPY) injection into the frontal cortex was examined in rats. NPY (1 micrograms/1 microliter) injected into the frontal cortex of rats with chronically implanted cannulae, increased their locomotor and exploratory activity in the open field test. Similar effects were observed after injection of the alpha-1-adrenergic agonist phenylephrine (PH) (3-19 micrograms/1 microliter). Behavioural stimulation after NPY or PH was totally abolished by pretreatment with benextramine (1.73-173 micrograms/1 microliter), a compound blocking NPY and alpha-adrenergic receptors. The NPY-induced activation of behaviour was also abolished by i.p. injection of prazosine (3 mg/kg), a specific alpha-1-adrenergic receptor antagonist. It is concluded that: 1) NPY injected into the rat frontal cortex induces an increase in the locomotor and exploratory activity of the animals; and 2) indirect activation of alpha-1-adrenergic receptor seems to play a crucial role in the observed behavioural effects.

Topics: Adrenergic alpha-Antagonists; Animals; Behavior, Animal; Cystamine; Frontal Lobe; Male; Motor Activity; Neuropeptide Y; Phenylephrine; Prazosin; Rats; Rats, Wistar; Receptors, Adrenergic, alpha

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.

Topics: Adrenergic alpha-Antagonists; Amino Acid Sequence; Animals; Cystamine; Male; Molecular Sequence Data; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Structure-Activity Relationship

In adult, sexually-experienced male rats, the NPY-antagonist benextramine--at the doses of 5 and 10 mg/kg i.p. and of 50 micrograms/rat i.c.v.--significantly and specifically improved several parameters of copulatory activity. These data may further support the idea that NPY plays a role in the complex regulation of male sexual function, seemingly at the brain level.

Topics: Animals; Cystamine; Female; Male; Neuropeptide Y; Rats; Rats, Wistar; Sexual Behavior, Animal

Analogs of N,N'-bis[6-[(2-methoxybenzyl)amino]hex-1-yl]cystamine (benextramine, BXT, 2) were synthesized using solution-phase peptide synthesis methodology and analyzed for activity in displacing specifically bound 1 nM N-[propionyl-3H]neuropeptide Y([3H]NPY) from benextramine-sensitive neuropeptide Y (NPY) binding sites in rat brain. Our new synthetic approach to these analogs began with the acylation of cystamine with the N-hydroxysuccinimide ester of tert-butyloxycarbonyl (t-Boc) protected 6-aminohexanoic acid, followed by deprotection of the t-Boc groups with 4 N HCl in dioxane. Acylation of this symmetric diamine with N-hydroxysuccinimide esters of appropriately substituted benzoic acids, followed by reduction of the resultant tetraamides with diborane in refluxing THF, afforded the target compounds. The BXT analog lacking the benzylic group (i.e., compound 11) had no [3H]NPY displacement activity at concentrations up to 1.4 x 10(-3) M. The 9-fold range in activities observed for the ortho, meta, and para regioisomers of the methoxy, chloro, and hydroxy benextramine analogs at benextramine-sensitive NPY rat brain binding sites does not differ from the range of potencies observed at alpha-adrenoceptors. However, the order of potencies at [3H]NPY sites differs from the order of potencies at alpha-adrenoceptors, with the m-methoxyphenyl (9a), m-hydroxyphenyl (10b), and 2-naphthyl (9f) analogs being the most active at [3H]NPY binding sites. The present results demonstrate the importance of the benzylic moiety for BXT's NPY antagonist activity, and suggest that the BXT binding site on the NPY receptor is significantly distinct from that on the alpha-adrenoceptor.

Topics: Adrenergic alpha-Antagonists; Amino Acid Sequence; Animals; Binding Sites; Brain; Cystamine; Drug Interactions; Male; Molecular Sequence Data; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Structure-Activity Relationship

The present study examined the effects of neuropeptide Y (NPY) and a selective NPY1 receptor agonist, leucine31 proline34 neuropeptide Y ([leu31,pro34]NPY) on gastric lesion formation and gastric secretion in three preparations: Basal gastric acid secretion in conscious rats, restraint-induced gastric lesion formation and acid and pepsin output and gastric mucosal damage in pylorus-ligated rats. The hypothesis that benextramine, a non-selective NPY receptor antagonist, could attenuate responses to NPY or [leu31,pro34]NPY was also tested. Both NPY and [leu31,pro34]NPY (i.p. and i.c.v.) decreased basal gastric acid output, restraint-induced gastric lesion formation, and acid and pepsin secretion and gastric mucosal damage in pylorus-ligated rats. The magnitude of inhibition of secretion and of ulcer reduction was significantly greater for [leu31,pro34]NPY than for NPY at comparable doses. Benextramine blocked the protective effect of NPY and [leu31,pro34]NPY against restraint-induced gastric mucosal injury. Both central and peripheral treatment with benextramine blocked the antisecretory effects of centrally administered NPY and [leu31,pro34]NPY. These data were consistent with both a central and a peripheral action of NPY on the gut, possibly through Y1 receptors.

Topics: Adrenergic alpha-Antagonists; Animals; Cystamine; Gastric Acid; Gastric Mucosa; Immobilization; Ligation; Male; Neuropeptide Y; Pepsin A; Pylorus; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Stomach; Stomach Ulcer; Stress, Physiological

Neuropeptide Y (NPY), a potent pressor agent and vasoconstrictor, is thought to contribute to the sympathetically mediated postsynaptic regulation of blood pressure primarily through activation of vascular Y1 rather than Y2 NPY receptors. However, data are available that conflict with this conclusion. In this article, the relative roles of postsynaptic Y1 and Y2 receptors as mediators of direct NPY-induced isolated rat femoral artery ring vasoconstriction were evaluated through use of selective Y1 and Y2 agonists, [Leu31, Pro34]NPY ([Leu, Pro]NPY) and NPY13-36 [NPY(13-36)], respectively, and the NPY receptor antagonist benextramine (BXT). NPY, [Leu, Pro]NPY and NPY(13-36) were equipotent as vasoconstrictors, and constriction induced by each peptide, but not by the calcium channel agonist BAY K 8644 (BAY), was almost completely blocked by 10 microM BXT. Each of the three peptides also induced self- and cross-desensitization and protection from BXT blockade, except that [Leu, Pro]NPY neither desensitized nor protected NPY(13-36)-associated responses. NPY also failed to protect [Leu, Pro]NPY- and NPY(13-36)-elicited constriction, and NPY(13-36) failed to provide self-protection, from BXT blockade. However, in these instances, as opposed to the [Leu, Pro]NPY-NPY(13-36) cross-protection experiments, the occurrence of protection was probably masked by the relatively large magnitude of desensitization concurrently induced by the protecting peptide. Taken together, the present findings suggest that NPY-induced rat femoral artery vasoconstriction is mediated by separate, BXT-sensitive, postsynaptic Y1 ([Leu, Pro]NPY-activated) and Y2 [NPY(13-36)-activated] receptors.

Topics: Animals; Cystamine; Drug Antagonism; Femoral Artery; In Vitro Techniques; Male; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Synapses; Vasoconstriction

Benextramine, a tetramine disulfide, irreversibly inhibits neuropeptide Y (NPY) binding to the 50-kDa Y2 NPY receptor in bovine hippocampus (Li, W., MacDonald, R. G., and Hexum, T. D. (1991) Eur. J. Pharmacol. 207, 89-91). Evidence is presented that this inhibition occurs through a thiol-disulfide exchange. Treatment of bovine hippocampal membranes with benextramine inhibited NPY affinity cross-linking to the 50-kDa receptor. This inhibition of labeling was not affected by washing the membranes, but could be completely reversed by the addition of several thiol reducing reagents, including reduced glutathione, beta-mercaptoethanol, and cysteine. Benextramine inhibited 70% of NPY-specific labeling and was much more effective than other sulfhydryl reactive agents, such as oxidized glutathione, cystamine, and 5,5'-dithio-bis(2-nitrobenzoic acid). Furthermore, the sulfhydryl-modifying agents N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid specifically decreased NPY affinity labeling. Finally, NPY labeling of the 50-kDa receptor was reduced by the heavy metal ions Zn2+, Cu2+, and Hg2+. Preincubation with NPY prevented Y2 receptors from being inactivated by either 400 microM N-ethylmaleimide or 1 mM benextramine. These results suggest that one or more benextramine-sensitive sulfhydryl groups on the Y2 receptor are important for NPY binding activity.

Topics: Adrenergic alpha-Antagonists; Affinity Labels; Animals; Cattle; Cystamine; Electrophoresis, Polyacrylamide Gel; Hippocampus; In Vitro Techniques; Membranes; Neuropeptide Y; Receptors, Neuropeptide Y; Receptors, Neurotransmitter; Sulfhydryl Compounds

Earlier investigation of the vascular actions of Neuropeptide Y (NPY) led us to propose that distinct receptors mediated the prejunctional inhibition of periarterial nerve-stimulated norepinephrine (NE) release and the postjunctional potentiation of the increase in perfusion pressure elicited by vasoconstrictors. These receptors were designated Y2 and Y1, respectively, based on the ability of C-terminal fragments to mimic the former action. The present study investigates further the involvement of these putative receptor subtypes in the isolated and perfused mesenteric arterial bed. [Leu31Pro34]NPY, a novel analog with specificity at the Y1 receptors, potentiated the increase in perfusion pressure elicited by exogenously administered NE and arginine vasopressin, confirming the existence of this NPY subtype postjunctionally. This immediate and prolonged potentiation was abolished by phentolamine, attenuated by benextramine and the reputed NPY antagonist, PYX1. [11-36]NPY also produced a concentration-dependent potentiation of NE-stimulated increase in perfusion pressure suggesting that the Y2 receptor subtype may also be present postjunctionally in this model of the vascular neuroeffector junction. The finding that the profile of this potentiation differed from that elicited by [Leu31Pro34]NPY and, in contrast to the latter, was not attenuated by PYX1, intimates the existence of both distinct subtypes postjunctionally. [Leu31Pro34]NPY also reduced periarterial nerve-stimulated release of NE with a concomitant reduction in perfusion pressure indicating, in addition to the Y2 subtype, the presence of the Y1 receptor prejunctionally in the rat mesenteric arterial bed.

Topics: Adrenergic alpha-Antagonists; Animals; Cystamine; Drug Interactions; Male; Mesenteric Arteries; Neuropeptide Y; Norepinephrine; Phentolamine; Rats; Rats, Inbred Strains; Receptors, Neuropeptide Y; Receptors, Neurotransmitter

Pre-incubation of rat brain membranes with 200 microM benextramine followed by extensive dilution and washing to remove unbound ligand reduced Bmax for N-[propionyl-3H]-NPY (3H-NPY) specific binding by 61% relative to control membranes treated identically but in the absence of benextramine. When rat brain membranes were co-incubated with 3H-NPY and 57 microM benextramine, there was a significant shift to the right; the apparent Kd for 3H-NPY binding increased two-fold relative to control membranes. These data are consistent with the hypothesis that benextramine is a competitive and irreversible ligand for a population (60-65%) of rat brain NPY binding sites. 'Paired tube' assays were then used to determine the selectivity of these benextramine-sensitive and insensitive 3H-NPY binding site populations. PYY, NPY and NPY13-36 each displaced 100% of 3H-NPY from rat brain membrane binding sites both in the absence and presence of 1 mM benextramine. In contrast, [Leu31,Pro34]NPY displayed the same binding site selectivity as benextramine in displacing 65% of 3H-NPY from specific binding sites on untreated rat brain membranes, and it failed to displace 3H-NPY from membranes treated with 1 mM benextramine. Thus the selectivity of the benextramine-insensitive 3H-NPY binding site population--PYY > = NPY > NPY13-36 >> [Leu31,Pro34]NPY--is characteristic of a Y2-like NPY binding site population, while the benextramine-sensitive 3H-NPY binding sites appear to be a Y1-like binding site population.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Brain; Cystamine; Male; Membranes; Molecular Sequence Data; Neuropeptide Y; Peptide YY; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Sensitivity and Specificity; Tritium

Affinity labeling of iodinated neuropeptide Y (NPY) to bovine hippocampal binding proteins revealed that benextramine inhibited specific NPY labeling of the 50 kDa NPY binding protein (Y2 binding protein) in a dose-dependent manner (IC50 = 33 microM). Hippocampal membranes, which were pretreated with benextramine and washed, exhibited decreased [125I]NPY labeling of binding proteins in a similar dose-dependent manner. These findings demonstrate that benextramine irreversibly blocks specific NPY binding to the 50 kDa NPY Y2 binding protein.

Topics: Affinity Labels; Animals; Carrier Proteins; Cattle; Cross-Linking Reagents; Cystamine; Hippocampus; In Vitro Techniques; Iodine Radioisotopes; Membranes; Neuropeptide Y; Succinimides

Norepinephrine (NE)-evoked vasoconstrictor and pressor responses are reduced after prolonged exposure; such desensitization is observed both clinically and experimentally. The vasoconstrictor neuropeptide Y (NPY) coexists with NE in perivascular sympathetic nerves, and the results of both in vivo and in vitro studies have indicated functional cooperation between NE and NPY. We propose that NPY becomes increasingly important in situations of high sympathetic activity associated with blunted NE responses. Prolonged NE infusion in conscious rats resulted in adrenergic desensitization; however, NPY administration restored the responsiveness to NE. In naive rats, NE greatly enhanced the pressor action of NPY. An analogous phenomenon was observed in the rabbit isolated pulmonary artery, which failed to respond to NPY unless preexposed to NE; this action of NE was only partly inhibited by conventional adrenoceptor and Ca2+ influx blockade. Conversely, NPY enhanced NE-evoked constriction, in particular when the alpha-adrenoceptor reserve was eliminated. It is proposed that threshold synergism, in part caused by converging stimulation of phospholipase C, accounts for much of the NPY/NE cooperativity. We conclude that 1) NPY and NE cooperate to produce vasoconstriction, both in vivo and in vitro; 2) NPY has the capacity to reverse adrenergic desensitization but not vice versa; 3) NE enhances NPY-evoked vasoconstriction, in part independently of conventional adrenoceptor blockade; 4) threshold synergism phenomena, but not "receptor-receptor interactions," account for (most of) the observed NPY/NE cooperation; and 5) when present, alpha-adrenoceptor reserve prevents the lowering of the NE threshold by NPY.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Consciousness; Cystamine; Drug Synergism; Inositol Phosphates; Male; Neuropeptide Y; Norepinephrine; Prazosin; Pulmonary Artery; Radioligand Assay; Rats; Rats, Inbred Strains; Vasoconstrictor Agents