Antihistaminic and antimuscarinic effects of amitriptyline on guinea pig ileal electrolyte transport and muscle contractility in vitro

Amitriptyline, a tricyclic antidepressant, was tested for antimuscarinic and antihistamine effects against bethanechol and histamine-stimulated contractility and secretion in the guinea pig ileum in vitro. Comparisons were made with muscarinic-receptor antagonists, as well as with H1 and H2 histamine-receptor antagonists. Amitriptyline (0.01-5.0 microM) produced a parallel rightward shift in the concentration-response curves to histamine in muscle (Ki 0.4 nM) and mucosa (Ki 450 nM). The H1-receptor antagonists pyrilamine and diphenhydramine were less potent against histamine in the muscle and more potent against histamine in the mucosa than was amitriptyline. The H2-receptor antagonist cimetidine was ineffective in the muscle and mucosa. Amitriptyline (0.1-2 microM) also produced a parallel rightward shift in the concentration-response curve to bethanechol in muscle (Ki 133 nM) and mucosa (Ki 143 nM). Against bethanechol, in both tissues, atropine and 4-diphenylacetoxy-N-methyl piperidine methiodide were more potent competitive antagonists than was amitriptyline. Pirenzepine produced a competitive blockade of bethanechol in the muscle and a noncompetitive blockade in the mucosa. The data indicate that amitriptyline exerts more potent antihistaminic effects on guinea pig ileal muscle than the mucosa but that the tricyclic drug is equipotent as an antimuscarinic in both tissues.     

Blocking action of terodiline on calcium channels in single smooth muscle cells of the guinea pig urinary bladder.

The blocking action of terodiline, a nonspecific organic Ca++ antagonist, on smooth muscle Ca++ channels of the guinea pig urinary bladder was investigated. Inward Ca++ currents were recorded from smooth muscle cells isolated from the urinary bladder using the whole-cell patch-clamp technique. In the absence of terodiline, a use-dependent reduction in the amplitude of inward Ca++ current was observed at a stimulus frequency of 0.2 Hz. When terodiline (1-10 microM) was applied, the use-dependent reduction was accelerated markedly, depending on the stimulus frequency. The blocking action of terodiline was also dose-dependent; the Kd value as measured at the end of 20 times repetitive stimulation at 0.2 Hz was 1.7 microM. In addition to such a use-dependent block, terodiline produced a hyperpolarizing shift in the steady-state inactivation curve. The results suggest that terodiline preferentially binds to the Ca++ channel in the open state and also in the inactivated state.     

Muscarinic receptors: relationships among phosphoinositide breakdown, adenylate cyclase inhibition, in vitro detrusor muscle contractions and in vivo cystometrogram studies in guinea pig bladder

The relationships between activation of muscarinic receptors in guinea pig bladder measured as carbachol-stimulated inositol phosphate (IP) accumulation, oxotremorine-induced adenylate cyclase (AC) inhibition and bladder detrusor smooth muscle contraction determined in vitro as well as in vivo in the slow filling cystometrogram (CMG), were analyzed from the potencies of a number of muscarinic antagonists to block these responses. Significant positive linear correlations were found among the inhibitory potencies of 10 muscarinic antagonists to inhibit phosphoinositide (PI) turnover and both detrusor muscle contraction in vitro, as well as peak intravesical bladder pressure in vivo in the CMG (r = 0.8, P less than .01). In contrast, there was no significant correlation between the potency of antagonists to block the AC inhibitory response and either in vitro or in vivo guinea pig bladder contractions (P greater than .05). Muscarinic agonists inhibited basal AC activity to a maximum of 20% in a GTP-dependent, Na+-sensitive manner and dose dependently stimulated both PI breakdown (3- to 4-fold) and isolated detrusor contractions. Again, a significant correlation (r = 0.9, P less than .01) was calculated among the potencies of seven muscarinic agonists to elicit PI turnover and in vitro muscle contraction, whereas no significant correlation was observed between their potencies to inhibit AC activity and contractile responses in vitro. Collectively, the data suggest that IP accumulation and presumably IP-induced Ca++ release may function as the transducing mechanism for cholinergic contraction of the urinary bladder.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile responses in bladder body, bladder neck and prostate from rat, guinea pig and cat

Lower urinary tract smooth muscle displays marked heterogeneity in pharmacologic responsiveness to contractile agents. The present study details differences among species with regard to muscarinic, adrenergic, histaminergic and serotonergic agonists in the bladder body, bladder neck and prostate from guinea pig, rat and cat. Under in vitro conditions, all smooth muscle preparations contracted to potassium chloride. The muscarinic agonist, carbamylcholine, produced maximal contraction, whereas alpha receptor agonists exerted only minimal, if any, effect in bladder body preparations from all three species. In contrast, alpha receptor-mediated responses predominated relative to muscarinic responses in bladder neck preparations from all three species. Prostatic contractility was examined in tissue from guinea pig and rat and contraction occurred to both alpha and muscarinic receptor agonists. Contractile response to norepinephrine in bladder neck and prostate was potentiated by neuronal uptake inhibition but not by beta receptor blockade. Serotonin and histamine exhibited more diverse effects among species and tissues. In general, histamine contracted all three tissues from guinea pig with minimal contraction occurring in tissues from rat or cat. On the other hand, serotonin markedly contracted the cat bladder body and rat prostate, but exerted no effect on tissues from the guinea pig. These data reinforce and detail the heterogeneity of pharmacologic contractile responses in lower urinary tract smooth muscle. Furthermore, the studies document the relative similarity among species in cholinergic and adrenergic responsiveness and the dissimilarity among species in serotonergic and histaminergic responsiveness of lower urinary tract smooth muscle.     

Functional selectivity of muscarinic receptor antagonists for inhibition of M3-mediated phosphoinositide responses in guinea pig urinary bladder and submandibular salivary gland

Binding and functional affinities of the muscarinic acetylcholine (mACh) receptor antagonists darifenacin, tolterodine, oxybutynin, and atropine were assessed in Chinese hamster ovary (CHO) cells expressing the human recombinant M2 (CHO-m2) or M3 (CHO-m3) receptors, and in guinea pig bladder and submandibular gland. In [N-methyl-3H]scopolamine methyl chloride binding studies in CHO cells, darifenacin displayed selectivity (14.8-fold) for the M3 versus M2 mACh receptor subtype. Oxybutynin was nonselective, whereas atropine and tolterodine were weakly M2-selective (5.1- and 6.6-fold, respectively). Antagonist functional affinity estimates were determined by the inhibition of agonist-induced [3H]inositol phosphate accumulation in CHO-m3 cells and antagonism of the agonist-induced inhibition of forskolin-stimulated cyclic AMP accumulation in CHO-m2 cells. Darifenacin was the most M3-selective antagonist (32.4-fold), whereas oxybutynin, atropine, and tolterodine exhibited lesser selectivity. Functional affinity estimates in guinea pig urinary bladder and submandibular salivary gland using indices of phosphoinositide turnover revealed that oxybutynin, darifenacin, and tolterodine each displayed selectivity for the response in the bladder, relative to that seen in the submandibular gland (9.3-, 7.9-, and 7.4-fold, respectively). In contrast, atropine displayed a similar affinity in both tissues. These data demonstrate that in bladder, compared with submandibular gland from a single species, the mACh receptor antagonists darifenacin, tolterodine, and oxybutynin display selectivity to inhibit agonist-mediated phosphoinositide responses. It is proposed that both responses are mediated via M3 mACh receptor activation and that differential functional affinities displayed by some, but not all, antagonists are indicative of the influence of cell background upon the pharmacology of the M3 mACh receptor.     

Protein kinase activity associated with the surface of guinea pig macrophages

Protein kinase activity has been detected associated with the outer surface of guinea pig peritoneal macrophages. Macrophages incubated with [gamma-32P]ATP incorporated 32P-phosphate into cell-associated proteins. Inorganic phosphate did not compete, nor could inorganic [32P]phosphate substitute as the phosphate donor, demonstrating that transfer of phosphate from ATP to protein is direct and extracellular. The macrophage-associated protein kinase was also shown to phosphorylate added acceptor protein (histone) and to be tightly associated with the cell surface. Thus, a new ectoenzyme, a protein kinase, has been detected in macrophages.     

Muscarinic actions of an N-methyl-N-2-bromoethylamino analog of oxotremorine (BR 401) in the mouse

The pharmacological effects of N-[4-(2-bromoethylmethylamino)-2-butynyl]-2-pyrrolidone (BR 401) were compared in the mouse with those of N-[4-(2-chloroethylmethylamino)-2-butynl]-2-pyrrolidone (BM 123) and oxotremorine. BR 401 was more toxic than oxotremorine and BM 123 when administered i.v. (LD50, 0.7 mumol kg-1), but less toxic than oxotremorine when given i.p. (LD50, 39 mumol kg-1). Atropine and methylatropine (10 mumol kg-1 i.p.) increased the LD50 value of BR 401, given i.v., 75- to 100-fold. Upon i.v. administration, BR 401 was 2- to 3-fold more potent than oxotremorine and 10 to 20 times more potent than BM 123 in producing central (tremor and analgesia) and peripheral (salivation) muscarinic effects. However, after i.p. administration BR 401 was 3-fold less potent than oxotremorine in eliciting tremor and analgesia. The aziridinium ion (BR 401A), formed by cyclization of BR 401, produced salivation but no tremor. These observations suggest that BR 401 when given i.v. penetrates effectively into the central nervous system where it cyclizes rapidly to the pharmacologically active aziridinium ion. In contrast, after i.p. administration a large proportion of BR 401 will cyclize before it can reach the central nervous system. BM 123, because of its slower cyclization, enters the central nervous system effectively also by the i.p. route. Thus, central potency of 2-haloethylamines such as BR 401 and BM 123 is critically dependent not only on the rate of cyclization, but also on the route of administration. The duration of tremor induced by BR 401 and BM 123 was considerably shorter than that induced by oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective binding of bladder muscarinic receptors in relation to the pharmacokinetics of a novel antimuscarinic agent, imidafenacin, to treat overactive bladder

The binding of orally administered imidafenacin, used to treat overactive bladders, to muscarinic receptors in rat tissue was characterized based on pharmacokinetics. The binding in six tissues including bladder tissue was measured using [N-methyl-(3)H] scopolamine methyl chloride ([(3)H]NMS). Pharmacokinetic parameters were estimated from measurements of the concentration of imidafenacin in serum, the bladder, and the submaxillary gland by liquid chromatography-mass spectrometry/mass spectrometry. The receptor binding affinity of imidafenacin in vitro was significantly lower in the bladder than submaxillary gland or colon. The oral administration of imidafenacin (0.79, 1.57, and 6.26 μmol/kg) was characterized by a more selective and longer-lasting binding to muscarinic receptors in the bladder than other tissues. Imidafenacin showed little binding to brain muscarinic receptors, consistent with its minor effect on the central nervous system. Pharmacokinetic data showed that orally administered imidafenacin was distributed at a higher concentration in the bladder than the serum or submaxillary gland of rats. After the intravesical instillation of imidafenacin, there was significant binding of muscarinic receptors in the bladder. Furthermore, a significant level of imidafenacin was detected in the urine of rats given a 1.57 μmol/kg concentration of this agent. The present study demonstrated that imidafenacin administered orally distributes predominantly to the bladder and exerts more selective and longer-lasting effect on the bladder than other tissues, such as the submaxillary gland, colon, and brain. Furthermore, the imidafenacin excreted in urine may play an important role in pharmacokinetic and pharmacological selectivity.     

Fine specificity of genetic regulation of guinea pig T lymphocyte responses to angiotensin II and related peptides

Guinea pig T lymphocyte responses to the octapeptide antigen angiotensin II (NH(2)-Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)-OH; AII) were examined using various synthetic peptide analogues and homologues. Each peptide antigen was assessed for immunogenicity and antigenicity in strain 2 and strain 13 guinea pigs as determined by in vitro T cell proliferative responses. The genetic control of T cell responses to these peptides was found to be highly specific and capable of distinguishing subtle differences in the antigens. For example, strain 2 guinea pigs responded to AII and were low responders to [Val(5)]-AII, whereas strain 13 animals responded to [Val(5)]-AII but not to AII. The genetic control in this case involved the difference of one methyl group between Val(5) and Ile(5). Differences in T cell responsiveness by strain 2 and strain 13 guinea pigs were also observed with analogues involving para substitutions on the phenyl ring of Tyr(4) and of Phe(8). However, the genetic regulation of T cell responses did not seem to be based on a single peptide residue. For example, removal of Asp(1) allowed strain 13 animals to respond to the Ile(5)-containing analogue, but eliminated responsiveness to the Val(5)-containing analogue. Thus, the first and fifth AII residues are both involved in the regulation of strain 13 T cell responses. Substitutions for Tyr(4) and Phe(8) suggested that the same residue may serve to alter the specificity of T cell responses in one strain, and determine responsiveness or unresponsiveness in the other strain. One of the most striking observations is that T cell responsiveness to the various AII analogues and homologues randomly fluctuates between strain 2 and strain 13 guinea pigs, and in general neither strain responds to the same peptide antigens. This suggests that strain 2 and strain 13 T cell responses are rarely directed against the same antigenic determinants, and that the T cell antigen-combining diversity is usually exclusive between these two strains. These results are discussed with respect to the specificity of Ir gene control and the relationship between Ir gene function and antigen recognition by T cells. Note added in proof: More recent experiments using a new lot of [Val(5)]- AII have indicated that [Val(5)]-AII-immune strain 2 T cells show significant stimulation with AII but remain relatively low responders with [Val(5)]-AII, as shown in Table I. The difference in priming for cross-reactivity for AII with the different lots of [Val(5)]-AII is at present unknown.     

A guinea pig hippocampal slice model of organophosphate-induced seizure activity

Extracellular recording techniques have been used in the guinea pig hippocampal slice preparation to investigate the electrophysiological actions of the organophosphate (OP) anticholinesterase soman. When applied at a concentration of 100 nM, soman induced epileptiform activity in the CA1 region in approximately 75% of slices. This effect was mimicked by the anticholinesterases paraoxon (1 and 3 microM), physostigmine (30 microM), and neostigmine (30 microM), thus providing indirect evidence that the epileptiform response was mediated by elevated acetylcholine levels. Soman-induced bursting was inhibited by the muscarinic receptor antagonists atropine (concentrations tested, 0.1-10 microM), telenzepine (0.03-3 microM), AF-DX116 [11-(2-[(diethylamino)methyl]-1-piperidinyl acetyl)-5,11-dihydro-6H-pyrido 92.b-b) (1,4)-benzodiazepin-6-one] (0.3-300 microM), and biperiden (0.1-10 microM) and by the benzodiazepine anticonvulsants diazepam (3-30 microM) and midazolam (3-30 microM), but it was not inhibited by the nicotinic antagonists mecamylamine (30 microM) and methyllycaconitine (300 nM). In contrast to soman-induced epileptiform activity, bursting induced by the K(+) channel blocker 4-aminopyridine (30 microM), the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (30 nM) or perfusion with low Mg(2+) buffer was insensitive to atropine (10 microM). The ability of muscarinic antagonists and benzodiazepines to inhibit soman-induced epileptiform activity is in accordance with the in vivo pharmacology of soman-induced seizures and suggests that the guinea pig hippocampal slice preparation may provide a useful tool for the evaluation of novel anticonvulsant therapies for the treatment of seizures related to OP poisoning.     

Analysis of preformed xenoreactive antibodies in the discordant guinea pig to rat model using a guinea pig fibroblast-like cell line

In the discordant guinea pig (gp) to rat model of xenotransplantation, circulating xenoreactive natural antibodies (XNA) recognizing gp antigens are usually determined by an ELISA using membrane extracts of gp platelets. We analysed the lung-derived, fibroblast-like cell line JH 4 to detect XNA by ELISA or immunoblot, which was compared to primary gp cells, i.e. platelets, liver- and spleen cells. All membrane extracts proved to be useful to detect rat XNA directed against gp antigens by ELISA. In general, IgM responses of Lewis or C6 deficient PVG rats (PVG/C6-) were higher as compared to IgG responses. However, we observed great inter-individual variabilities. The strongest IgM response of Lewis rat sera was observed when the JH 4 cell line or gp liver cells were used as antigen. JH 4 cells also showed the strongest xenoreactivity with sera from PVG/C6- rats. These data demonstrate that JH 4 cells prove useful as antigen source for XNA ELISA. In immunoblot, individual sera of the two different rat strains showed the same antigen patterns using a gp membrane extract of one particular cell type. However, the different gp cell types showed a distinct pattern of antigen expression. Whereas the JH 4 cells, platelets and spleen cells express xenoreactive proteins of the same size, a unique pattern of proteins was detected in liver cells.     

Analysis of preformed xenoreactive antibodies in the discordant guinea pig to rat model using a guinea pig fibroblast-like cell line

In the discordant guinea pig (gp) to rat model of xenotransplantation, circulating xenoreactive natural antibodies (XNA) recognizing gp antigens are usually determined by an ELISA using membrane extracts of gp platelets. We analysed the lung-derived, fibroblast-like cell line JH 4 to detect XNA by ELISA or immunoblot, which was compared to primary gp cells, i.e. platelets, liver- and spleen cells. All membrane extracts proved to be useful to detect rat XNA directed against gp antigens by ELISA. In general, IgM responses of Lewis or C6 deficient PVG rats (PVG/C6-) were higher as compared to IgG responses. However, we observed great inter-individual variabilities. The strongest IgM response of Lewis rat sera was observed when the JH 4 cell line or gp liver cells were used as antigen. JH 4 cells also showed the strongest xenoreactivity with sera from PVG/C6- rats. These data demonstrate that JH 4 cells prove useful as antigen source for XNA ELISA. In immunoblot, individual sera of the two different rat strains showed the same antigen patterns using a gp membrane extract of one particular cell type. However, the different gp cell types showed a distinct pattern of antigen expression. Whereas the JH 4 cells, platelets and spleen cells express xenoreactive proteins of the same size, a unique pattern of proteins was detected in liver cells.     

Functional characterization of adenosine receptors and coupling to ATP-sensitive K+ channels in Guinea pig urinary bladder smooth muscle

Although multiple adenosine receptors have been identified, the subtype and underlying mechanisms involved in the relaxation response to adenosine in the urinary bladder remain unclear. The present study investigates changes in the membrane potential, as assessed by fluorescence-based techniques, of bladder smooth muscle cells by adenosine receptor agonists acting via ATP-sensitive potassium (K(ATP)) channels. Membrane hyperpolarization evoked by adenosine and various adenosine receptor subtype-selective agonists was attenuated or reversed by the K(ATP) channel blocker glyburide. Comparison of adenosine receptor agonist potencies eliciting membrane potential effects showed a rank order of potency 5'-N-ethyl-carboxamido adenosine (NECA; -log EC50 = 7.97) approximately 2-p-(2-carboxethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680; 7.65) > 2-chloro adenosine (5.90) approximately 2-chloro-N6-cyclopentyladenosine (CCPA; 5.51) approximately N6-cyclopentyladenosine approximately N6-(R)-phenylisopropyladenosine > 2-chloro- N6-(3-iodobenzyl)-adenosine-5'-N-methyl-carboxamide (2Cl-IBMECA; 4.78). Membrane potential responses were mimicked by forskolin, a known activator of adenylate cyclase, and papaverine, a phosphodiesterase inhibitor. The A(2A)-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), and the adenylate cyclase inhibitor N-(cis-2-phenyl-cyclopentyl) azacyclotridecan-2-imine-hydrochloride (MDL-12330A) inhibited the observed change in membrane potential evoked by adenosine and adenosine-receptor agonists. The rank order potency for relaxation of K+-stimulated guinea pig bladder strips, NECA (-log EC50 = 6.41) approximately CGS-21680 (6.38) > 2-chloro adenosine (5.90) > CCPA approximately 2Cl-IBMECA (>4.0) was comparable to that obtained from membrane potential measurements. Collectively, these studies demonstrate that adenosine-evoked membrane hyperpolarization and relaxation of bladder smooth muscle is mediated by A(2A) receptor-mediated activation of K(ATP) channels via adenylate cyclase and elevation of cAMP.     

The activity of gamma-glutamyltransferase after bile duct ligation in guinea pig.

The activity of gamma-glutamyltransferase was studied in guinea pig after bile duct ligation. In serum, an abrupt increase in activity up to 10--20 times the normal value was found 3 h after obstruction and the mean activity over the first 3 days following the operation was some 8 times the normal value. In liver, however, a small decline in activity could be demonstrated. The administration of cycloheximide did not influence the acute increase in serum activity. Bile duct ligation caused marked increases in serum bile acid levels which initially paralleled the serum gamma-glutamyltransferase activity. It is suggested that the increased serum activity may arise from the solubilization by bile acids of liver membrane-bound enzyme.     

A test for antinociceptive activity of narcotic and narcotic antagonist analgesics in the guinea pig.

Ethylenediamine tetraacetic acid (EDTA) was used as a nociceptive stimulus intradermally in the guinea pig. The responses evoked by EDTA included vocalization, biting and scratching at the site of injection and escape behavior. Nociception by EDTA was shown to be the result of its cation chelating activity. The suppression of EDTA-induced responses proved to be a rapid and effective antinociceptive test. The narcotic analgesics morphine, codeine, meperidine and methadone and the narcotic antagonist analgesics cyclazocine, cyclorphan, nalorphine and pentazocine were active. The slopes of the dose-response curves of the narcotic antagonist analgesics were significantly shallower than those of the narcotic analgesics. The test was highly specific for these analgesics. Antipyretic analgesics and various nonanalgesic drugs were inactive.     

Characterization of the class I antiarrhythmic activity of cibenzoline succinate in guinea pig papillary muscle

The effects of cibenzoline on transmembrane action potentials were examined in guinea pig papillary muscle. Cibenzoline (1-128 microM) did not alter the action potential durations at 50 and 90% of repolarization, the effective refractory period or the ratio of effective refractory period to action potential duration at 90% of repolarization. Likewise, the maximum diastolic potential was virtually unaffected. Cibenzoline depressed the maximum rate of rise of phase 0 (dV/dtmax). This effect was dependent on the rate of stimulation and occurred at a relatively low concentration (2 microM). The onset of use-dependent depression was monoexponential and dependent on the drug concentration, as well as the rate of stimulation. The rate of recovery from use-dependent depression also followed a single exponential time course but was independent of drug concentration and stimulation rate. When cibenzoline and lidocaine were combined in the tissue bath, dV/dtmax recovered with a double exponential time course. The first and second components of this recovery corresponded to the time course observed with lidocaine (first) and cibenzoline (second) alone. Also, the magnitude of the second component was less with the combination than with cibenzoline alone, indicating an interaction between the two drugs. In addition, cibenzoline shifted the curve relating normalized dV/dtmax to membrane potential in the hyperpolarizing direction. Finally, cibenzoline did not alter slow-response action potentials induced by elevated [K]o and isoproterenol. The authors conclude that cibenzoline acts as a class la antiarrhythmic agent in guinea pig papillary muscle.