Interaction between neurotransmitters and exogenous norepinephrine in isolated rat anococcygeus muscle.

Continuous electrical field stimulation (EFS) elicited a sustained contraction and significantly increased the EC50 value of norepinephrine (NE), shifting the concentration-response curve for NE to the right. Tetrodotoxin significantly reduced the continuous EFS-evoked increases in basal tone and produced further dextral shift in the concentration-response curve for NE. N-methylhydroxylamine and NG-monomethyl-L-arginine (L-NMMA) attenuated the "dual" effects of continuous EFS on NE-induced contractions. L-Arginine partially reversed the inhibitory effect of L-NMMA. The results of the present study suggest that continuous EFS causes simultaneous release of both excitatory and inhibitory neurotransmitters, and the interaction (or functional antagonism) between the inhibitory neurotransmitter (endogenous nitric oxide, NO) and the excitatory neurotransmitter (endogenous NE), as well as exogenous NE, may occur at postjunctional site(s).

Effects of omega-conotoxin GVIA on electrical field stimulation- and agonist-induced changes in cytosolic Ca2+ and tension in isolated rat anococcygeus muscle.

1. It has been reported that omega-conotoxin GVIA (omega-CgTx) blocks L- and N-type voltage-sensitive Ca2+ channels (VSCCs) in neurones and inhibits neurotransmitter release in various tissues. The present study investigates the effects of omega-CgTx on electrical field stimulation (EFS)- and agonist-induced changes in free cytosolic Ca2+ ([Ca2+]cyt) levels and tension in isolated fura-2 loaded rat anococcygeus muscle. 2. EFS produced frequency-dependent increases in [Ca+]cyt levels and contractions. Phentolamine (1 microM) and omega-CgTx (0.1 microM) significantly inhibited EFS-induced responses and shifted the frequency-response curves to the right. 3. alpha-adrenoceptor agonists (noradrenaline and clonidine) and carbachol (in the presence of phentolamine) produced concentration-dependent increases in [Ca2+]cyt levels and contractions. Though omega-CgTx (0.1 microM) significantly inhibited the increases in [Ca2+]cyt levels induced by low doses of noradrenaline, the overall concentration-response curves of [Ca2+]cyt and contractions for noradrenaline, clonidine, and carbachol were not affected by omega-CgTx. 4. When the tone of rat anococcygeus muscle was raised with either clonidine (0.1 microM) or carbachol (30 microM, in the presence of 3 microM phentolamine), EFS (2 Hz) produced reproducible decreases in [Ca2+]cyt levels and relaxations. These responses were significantly inhibited by omega-CgTx when the tissue was precontracted with clonidine, but not when it was precontracted with carbachol. 5. The results of the present study suggest that in rat anococcygeus muscle, omega-CgTx inhibits the EFS-induced release of both excitatory and inhibitory neurotransmitters, probably by blocking Ca2+ channels on nerve terminals. Furthermore, the Ca2+ channels present on the smooth muscle cell membrane, which are involved in the agonist-induced Ca2+ influx and contractions, may not be sensitive to omega-CgTx.

Analysis of bradykinin-induced relaxations in the rat isolated anococcygeus muscle.

The present study investigates the mechanism(s) of action of relaxations induced by bradykinin and by electrical field stimulation (EFS) in isolated rat anococcygeus muscle, where contractile tone has been elevated with clonidine. Bradykinin, EFS, and the bradykinin B1 receptor agonist, des-Arg9-bradykinin, produced quantitatively and qualitatively similar relaxations. Bradykinin B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (1 microM), attenuated the relaxation responses of bradykinin B1 receptor agonist and inhibited bradykinin and EFS-induced relaxation responses. Bradykinin B2 receptor antagonist, [beta-(2-thienyl)-Ala5,8,D-Phe7]-bradykinin (1 microM), significantly inhibited the relaxation responses of bradykinin, EFS, and bradykinin B1 receptor agonist. Methylene blue (30 microM) and N-methylhydroxylamine (1 mM) significantly inhibited the bradykinin- and EFS-induced relaxation responses. The relaxation responses of bradykinin and EFS were not affected by captopril (5 microM), superoxide dismutase (100 U/ml), and catalase (100 U/ml). Nitric oxide synthase inhibitor, L-NG-nitro-arginine (L-NOARG, 30 microM), significantly inhibited the EFS- and bradykinin-induced relaxation responses. L-arginine (100 microM) reversed the inhibitory effect of L-NOARG on the relaxation responses of EFS and bradykinin. In addition, L-arginine potentiated the relaxation responses of EFS and bradykinin. The data of the present study suggests that bradykinin, similar to EFS, generates an endogenous nitrate, probably nitric oxide, which subsequently activates guanylate cyclase and relaxes the rat anococcygeus muscle.

Opioid receptor activity of GI 87084B, a novel ultra-short acting analgesic, in isolated tissues.

GI 87084B (3-[4-methoxycarbonyl-4-[(1-oxopropyl) phenylamino]1-piperidine]propanoic acid, methyl ester, hydrochloride) was found to be a potent opioid agonist in the guinea pig ileum (EC50 = 2.4 +/- 0.6 nM), the rat vas deferens (EC50 = 387 +/- 44 nM) and the mouse vas deferens (EC50 = 39.5 +/- 7.4 nM). In the guinea pig ileum, GI 87084B, was roughly equivalent in potency to fentanyl (EC50 = 1.8 +/- 0.4 nM). GI 87084B was more potent in this tissue than alfentanil (EC50 = 20.1 +/- 1.2 nM) and less potent than sufentanil (EC50 = 0.3 +/- 0.09 nM). Schild analyses of antagonism of GI 87084B by naloxone yielded pKB values of 8.2 and slopes indistinguishable from unity in the guinea pig ileum and the mouse vas deferens. Insurmountable antagonism of GI 87084B by naloxone was observed in the rat vas deferens. However, an empirical measure of antagonist potency could be made: apparent pA2 = 8.1. The agonist dissociation constant (KA) for GI 87084B (220 +/- 90 nM) was determined by receptor alkylation with beta-chlornaltrexamine in the guinea pig ileum. Calculation of receptor occupancy suggested poor receptor-effector coupling and limited receptor reserve in the rat vas deferens, which could explain the insurmountable antagonism seen with higher concentrations of naloxone. These data suggest that GI 87084B acted through the mu class of opioid receptors to inhibit contraction induced by field stimulation in these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis, and pharmacological evaluation of ultrashort- to long-acting opioid analgetics.

In an effort to discover a potent ultrashort-acting mu opioid analgetic that is capable of metabolizing to an inactive species independent of hepatic function, several classes of 4-anilidopiperidine analgetics were synthesized and evaluated. One series of compounds displayed potent mu opioid agonist activity with a high degree of analgesic efficacy and an ultrashort to long duration of action. These analgetics, 4-(methoxycarbonyl)-4-[(1-oxopropyl)phenylamino]-1-piperidinepropanoi c acid alkyl esters, were evaluated in vitro in the guinea pig ileum for mu opioid activity, in vivo in the rat tail withdrawal assay for analgesic efficacy and duration of action, and in vitro in human whole blood for their ability to be metabolized in blood. Compounds in this series were all shown to be potent mu agonists in vitro, but depending upon the alkyl ester substitution the potency and duration of action in vivo varied substantially. The discrepancies between the in vitro and in vivo activities and variations in duration of action are probably due to different rates of ester hydrolysis by blood esterase(s). The SAR with respect to analgesic activity and duration of action as a function of the various esters synthesized is discussed. It was also demonstrated that the duration of action for the ultrashort-acting analgetic, 8, does not change upon prolonged infusion or administration of multiple bolus injections.

Analysis of the presence of postjunctional alpha-2 adrenoceptors in the rat anococcygeus muscle.

When the tone is raised by guanethidine, rat anococcygeus muscle produces inhibitory responses to field stimulation, whose mechanism is not understood properly. The present study is an attempt to investigate the role of alpha adrenoceptors in the field stimulation-induced relaxations in isolated rat anococcygeus muscle. When the tissues are contracted with clonidine, UK-14,304 and low doses of oxymetazoline, field stimulation produced relaxations at lower frequencies, but not in the tissues precontracted with phenylephrine and norepinephrine. Relaxations induced by low frequencies were blocked by idazoxan, but not by phentolamine, prazosin, indomethacin, N-methyl-hydroxylamine, ouabain or 3,4-diminopyridine. When the tone of the muscle is raised by norepinephrine, prazosin reversed the field stimulation-induced contractions to relaxation responses. The data of the present study suggested the possible involvement of alpha-2 adrenoceptors during the field stimulation-induced relaxations of the rat anococcygeus muscle. To analyze and quantitate the alpha-2 adrenoceptor antagonism in the rat anococgygeus muscle, Schild analyses of clonidine-induced contractions against idazoxan were conducted either for idazoxan alone or after partially alkylating the alpha-1 adrenoceptors with phenoxybenzamine and by pharmacologic resultant analysis by blocking the alpha-1 adrenoceptors with prazosin. The Schild regression for idazoxan and pharmacologic resultant analysis suggested that the rat anococcygeus muscle responds to alpha-2 agonists with alpha-1-mediated contractions and idazoxan competes with alpha-1 antagonists for the same site, i.e., alpha-1 adrenoceptor site. However, the atypical Schild regression of idazoxan after partial alkylation with phenoxybenzamine indicated the existence of a second alpha adrenoceptor site in the rat anococcygeus muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

A new method for estimation of agonist dissociation constants (KA): directly fitting the postinactivation concentration-response curve to a nested hyperbolic equation.

The common method for estimating agonist dissociation constants (KA) is the method proposed separately by Furchgott and Mackay. Concentrations of the given agonist producing the same response before ([A]) and after ([A']) irreversible inactivation of a fraction of the receptors (1-q) are described by the equation: 1/[A] = 1/(q*[A']) + (1-q)/(q*KA) and plotted on axes of 1/[A] vs. 1/[A']. The double reciprocal method suffers from the disadvantage that undue weight may be placed on values generated from the smallest observed responses. Our new method of estimating KA and q uses hyperbolic functions to directly fit both concentration-response curves. The control curve is fit to the logistic equation: response = (M* [A]n)/(kn + [A]n); where M is the maximal tissue response, n is the apparent kinetic order of the response at low [A] and k is [A] required for a half-maximal response. The postinactivation concentration-response curve is simultaneously fit to the following equation: response' = M/(((k/(q*KA*[A']))*(KA + [A']* (1-q)))n + 1). This new method was shown to determine KA and q more accurately and precisely than other methods when applied to an artificial data set. In experiments with the rat anococcygeus muscle, the nested hyperbolic method gave estimates of KA with less variance and less range than the double reciprocal method. The nested hyperbolic method was shown to be a valid method of estimating KA and q that has advantages over the other methods.

Analysis of putative alpha-1s adrenoceptor agonism by Sgd 101/75 in the rat anococcygeus muscle.

Sgd 101/75 [2-(2-methylindazol-4-imino)-imidazolidine hydrochloride] is an alpha 1 adrenoceptor agonist that shows a greater sensitivity to receptor alkylation with phenoxybenzamine (POB) than norepinephrine (NE) (present study). J. Coates, D.G. Weetman and their co-workers have concluded that Sgd 101/75 is selective for a subtype of alpha-1 receptors, i.e., alpha-1s. The present studies have shown that Sgd 101/75 was a full agonist in the rat anococcygeus muscle and acts at alpha-1 adrenoceptors, as determined by Schild analysis of prazosin antagonism (pKB = 9.24 +/- 0.21, slope = 0.87 +/- 0.13). Alkylation experiments showed that the contractile effects of Sgd 101/75 were more sensitive to POB than the effects of NE. These experiments also allowed the determination of KA values for these agonists (Sgd 101/75 KA = 1.5 +/- 0.6 X 10(-5) M, NE KA = 2.6 +/- 1.0 X 10(-6) M). At 3 X 10(-8) M POB (10 min), the contractile effect of Sgd 101/75 was eliminated, whereas the concentration-effect curve for NE was shifted approximately one log unit to the right with a 20% decrease in maximal response. After similar POB treatment, incubation with Sgd 101/75 (60 min, 3 X 10(-6) to 3 X 10(-4) M) shifted the NE curve to the right in a concentration-dependent manner. Schild regression of these data yielded a pKB = 5.22 +/- 0.09 and slope = 0.87 +/- 0.08. This pKB (-log KB) showed that Sgd 101/75 was competing for the same receptor after POB treatment and before POB treatment (mean of -log KA = 4.92 +/- 0.12).(ABSTRACT TRUNCATED AT 250 WORDS)

Naloxazone treatment in the guinea pig ileum in vitro reveals second functional opioid receptor site.

Guinea pig ilea were incubated in vitro with naloxazone, an irreversible opioid receptor antagonist, in an attempt to determine a dissociation constant (KA) value for the opioid agonist, BW 942C, by the method of partial receptor alkylation. However, concentrations of naloxazone up to 3 X 10(-4) M (120 min incubation-60 min washout) did not depress the maximal response or the slope of the concentration-response curve for BW 942C and, therefore, did not allow calculation of KA value. To analyze the residual activity of BW 942C, tissues were incubated with naloxone for 60 min after naloxazone treatment. Schild analysis of naloxone antagonism after 3 X 10(-6) M naloxazone produced a pKB of 8.0 +/- 0.06 and a slope of 0.88 +/- 0.03, suggesting simple competitive antagonism at a single site. In the absence of naloxazone treatment, naloxone 10(-8) to 10(-5) M antagonized the effects of BW 942C yielding a pKB value and slope of 8.34 +/- 0.08 and 1.0 +/- 0.04, respectively. Schild analysis of naloxone antagonism after 10(-4) M naloxazone yielded a pA2 of 6.23 +/- 0.20 and a slope of 0.55 +/- 0.08. These values were not consistent with simple competitive antagonism at a single receptor site. Modeled curves showed that the data for naloxone antagonism after 10(-4) M naloxazone were consistent with action at two receptor sites with naloxone pKB values of approximately 8.3 (experimentally determined) and approximately 6.0. An alternative explanation of altered affinity for BW 942C and naloxone at a single site produced by naloxazone treatment cannot be excluded.

Quantitative assessment of the pre- and postsynaptic alpha adrenoceptor antagonist potency of amitriptyline.

The pre- and postsynaptic alpha adrenoceptor blocking affinity of amitriptyline was determined in isolated tissues by Schild regression analysis. In the absence of uptake-1 blockade with cocaine, amitriptyline treatment (3 X 10(-8)-3 X 10(-6) M) affected only marginally norepinephrine concentration-response curves in the rat anococcygeus muscle; a result suggesting opposing pharmacological effects (uptake-1 blockade and alpha blockade). After cocaine (3 X 10(-5) M) treatment, amitriptyline (3 X 10(-8)-3 X 10(-6) M) antagonized competitively concentration-response curves to norepinephrine, yielding a postsynaptic pKb of 7.51. A similar pKb was obtained when methoxamine was the agonist. Presynaptic alpha blocking affinity was determined by using the field-stimulated rat vas deferens. Stimulus conditions were chosen which minimized the inhibition of twitch height by high concentrations of cocaine. Using these conditions (10 Hz, 200 msec duration at 100-sec intervals), amitriptyline antagonized competitively clonidine inhibition of field-stimulated twitch contractions, yielding at pKb of 5.23. The presynaptic pKb was not changed in the presence of theophylline (10(-4) M). Amitriptyline was also observed to increase the release of [3H]norepinephrine from the field-stimulated rat anococcygeus muscle pretreated with cocaine. Although this effect primarily reflects alpha blockade, other biochemical and presynaptic mechanisms may be involved. Comparing the pre- and postsynaptic alpha blocking affinities indicates that amitriptyline has 191 X greater affinity for post- than presynaptic alpha adrenoceptors (i.e., alpha-1 much greater than alpha-2). The relevance of these observations to the mechanism of action and side effects of amitriptyline are discussed.

Enhancement of methacholine-stimulated guanosine 3':5'-cyclic monophosphate formation in supersensitive guinea pig vasa deferentia.

Decentralization of the guinea pig vas deferens resulted in supersensitivity of the contractile response to methacholine. Methacholine also elevated cyclic GMP to a greater extent in the decentralized vas deferens than in sham-operated controls. Decentralization did not alter basal cyclic AMP or cyclic GMP content. These observations are analogous to the enhanced responsiveness of cyclic AMP formation in supersensitive beta adrenergic systems.