Pig Ileal Longitudinal Smooth Muscle Research Articles

Reversal of multidrug resistance in cancer cells by novel asymmetrical 1,4-dihydropyridines

Multidrug resistance (MDR) is an important obstacle that limits the efficacy of chemotherapy in many types of cancer. In this study, 14 novel asymmetrical DHPs possessing pyridyl alkyl carboxylate substitutions at C3 and alkyl carboxylate groups at C5 in addition to a nitroimidazole or nitrophenyl moiety at C4 position were synthesized. Calcium channel blocking (CCB) activity was measured in guinea pig ileal longitudinal smooth muscle. Cytotoxicity was tested on 4 human cancer cell lines, while MDR reversal capacity was examined on P-glycoprotein overexpressing doxorubicin resistant MES-SA-DX5 and compared with non-resistant MES-SA cells. Compounds showed different CCB (IC50: 29.3 nM-4.75 μM) and cytotoxic activities (IC50: 6.4 to more than 100 μM). Several compounds having nitrophenyl moiety at C4, could significantly reverse resistance to doxorubicin at 0.5 and 1 μM. The most active ones were 7e and 7g containing ethyl carboxylate and isopropyl carboxylate at C5, respectively. CCB activity, which is considered an undesirable effect for these agents, of 7e and 7g were 33 and 20 times lower than nifedipine, respectively. In conclusion, the newly synthesized asymmetrical DHP compounds showed promising MDR reversal and antitumoral activities with low CCB effects and could be of therapeutic value in drug resistant cancer.

Radiosynthesis of dimethyl-2-[18F]-(fluoromethyl)-6-methyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate for L-type calcium channel imaging

Abstract Dimethyl 2-(fluoromethyl)-6-methyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate 4a, a fluorinated nifedipine analog, has been shown to elicit significant calcium channel blocker activity using a guinea pig ileal longitudinal smooth muscle model. In order to perform biological studies for detection of L-type calcium channel distribution, we decided to prepare the [ 18F]-labeled compound. The latter compound was prepared in no-carrier-added (n.c.a.) form from dimethyl 2-(bromomethyl)-6-methyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate 2 in one step at 80 °C in Kryptofix[222]/K[ 18F]F and acetonitrile as a solvent in 15 min. Column chromatography afforded the radiochemically pure compound in 20 min. Radiochemical purity of the 18F-nifedipine was determined by RTLC and HPLC (>98%) and specific activity of 21–48 GBq/μmol (EOB).

Synthesis of alkyl 6‐methyl‐4‐(2‐pyridyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates for evaluation as calcium channel antagonists

Abstractmagnified imageThe Bigenelli acid catalyzed condensation of 2‐pyridylcarboxaldehyde (1), urea (2) and an alkyl acetoacetate (3) afforded the respective alkyl (Me, Et, i‐Pr, i‐Bu, t‐Bu) 6‐methyl‐4‐(2‐pyridyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates (4a‐e). The most potent calcium channel antagonist ethyl 6‐methyl‐4‐(2‐pyridyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylate (4b, IC50 = 1.67 × 10−5 M) wasa much weaker calcium channel antagonist than the reference drug nifedipine (Adalat®, IC50 = 1.40 × 10−8 M) on guinea pig ileal longitudinal smooth muscle (GPILSM). The alkyl 6‐methyl‐4‐(2‐pyridyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates did not show any inotropic effect on heart since no increase, or decrease, in the contractile force of guinea pig left atrium was observed. These structure activity studies show that the alkyl 6‐methyl‐4‐(2‐pyridyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates (4a‐e) are partial bioisosteres of nifedipine with respect to calcium channel antagonist activity on guinea pig ileal longitudinal smooth muscle (GPILSM).

Synthesis of alkyl 6‐methyl‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates possessing a N‐3 nitro substituent to determine calcium channel modulation structure‐activity relationships

Abstractmagnified imageThe Bigenelli acid catalyzed condensation of 2‐trifluoromethylbenzaldehyde (1), urea (2) and an alkyl acetoacetate (3) afforded the respective alkyl (Me, Et, i‐Pr, i‐Bu) 6‐methyl‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylate (4‐7). Subsequent N3‐nitration of the alkyl esters (4‐7) using Cu(NO3)2 3H2O and Ac2O furnished the target alkyl 6‐methyl‐3‐nitro‐4‐(2‐trifluoromethylphenyl)‐1,2,3,4‐tetrahydro‐2H‐pyrimidine‐2‐one‐5‐carboxylates (8‐11). The N3‐nitro compounds (8‐11) were less potent calcium channel antagonists (IC50 values in the 1.9 × 10−7 to 3.9 × 10−6 M range) on guinea pig ileal longitudinal smooth muscle than the reference drug nifedipine (Adalat®, IC50 = 1.4 × 10−8 M). In vitro calcium channel modulation studies on guinea pig left atrium (GPLA) showed that the methyl and ethyl esters (8‐9) induced a weak‐to‐modest positive inotropic (agonist) effect, and that the inactive isopropyl (10) and isobutyl (11) esters did not alter the cardiac contractile force of GPLA.

Design, Synthesis, and Calcium Channel Antagonist Activity of New 1,4‐Dihydropyridines Containing 4‐(5)‐Chloro‐2‐ethyl‐5‐(4)‐imidazolyl Substituent

A series of dialkyl, dicycloalkyl, and diaryl ester analogues of nifedipine, in which the ortho-nitro phenyl group at position 4 is replaced by the 4-(5)-chloro-2-ethyl-5-(4)-imidazolyl substituent, were synthesized and evaluated as calcium channel antagonists using the high K+ contraction of guinea pig ileal longitudinal smooth muscle. The results for the symmetrical ester series showed that increasing the length of the chain in C3- and C5-ester substituents increased the activity and the most active compound was the diphenylethyl ester derivative, so it was more active than the reference drug nifedipine. In unsymmetrical diester series, when R1 is methyl or ethyl, increasing lipophilic properties in the R substituent, increased the activity. The most active compounds were methyl/phenethyl and ethyl/phenethyl ester derivatives, being slightly more active than nifedipine.

Hantzsch 1,4-dihydropyridines containing a nitrooxyalkyl ester moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of 3-nitrooxyalkyl 5-alkyl 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates were prepared using a modified Hantzsch reaction that involved the condensation of a nitrooxyalkyl acetoacetate with an alkyl 3-aminocrotonate and a pyridinecarboxaldehyde. 1H NMR nuclear Overhauser enhancement (nOe) studies for 3-(3-nitrooxypropyl) 5-isopropyl 1,4-dihydro-2,6-dimethyl-4-(2-pyridyl)-3,5-pyridinedicarboxylate (17) indicates a predominant rotamer exists in solution where the pyridyl nitrogen atom is orientated above the 1,4-DHP ring system, and the pyridyl nitrogen atom is antiperiplanar to the 1,4-DHP ring H-4 proton. Variable temperature 1H NMR studies (–30 to +60°C) showed the 1,4-DHP NH proton in 17 is H-bonded in CHCl3 solution. This interaction is believed to be due to intermolecular H-bonding between the pyridyl nitrogen free electron pair and the 1,4-DHP NH proton. In vitro calcium channel antagonist (CCA) activities were determined using a muscarinic-receptor-mediated Ca+2-dependent contraction of guinea pig ileal longitudinal smooth muscle assay. This class of compounds exhibited lower CCA activity (IC50 = 5.3 × 10–6 to 3.5 × 10–8 M range) than the reference drug nifedipine (IC50 = 1.4 × 10–8 M). For compounds having C-3 –CH2CH2ONO2 and C-4 pyridyl substituents, the C-5 alkyl was a determinant of CCA (i-Pr > the approximately equipotent i-Bu, t-Bu, and Et analogs). The point of attachment of the isomeric C-4 pyridyl substituent was a determinant of CCA when C-3 –CH2CH2ONO2 and C-5 i-Pr substituents were present providing the potency profile 2-pyridyl ≥ 3-pyridyl > 4-pyridyl. CCA with respect to the C-3 nitrooxyalkyl substituent was inversely dependent on the length of the alkyl spacer. The percent nitric oxide (·NO) released in vitro by this group of compounds (range of 0.03–0.43%/ONO2 group), quantified as nitrite by reaction with the Griess reagent, was lower than that for the reference drug glycerol trinitrate (3.81%/ONO2 group). Nitric oxide release studies showed that the %·NO released was dependent on the number of ONO2 groups/molecule. A QSAR study for this group of compounds showed a correlation between the specific polarizability descriptor (SpPol) and %·NO release. Drug Dev. Res. 51:233–243, 2000. © 2001 Wiley-Liss, Inc.

Design and syntheses of C‐4 4‐(1‐methoxycarbonyl‐1,2‐dihydropyridyl)‐, and 3‐[1‐methoxycarbonyl‐1,2‐(and 1,6‐)dihydropyridyl]‐, derivatives of alkyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐5‐pyridinecarboxylates with cardiospecific calcium channel agonist activity

A group of hitherto unknown alkyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(dihydropyridyl)-5-pyridinecarboxylates possessing 4-[4-(1-methoxycarbonyl-1,2--dihydropyridyl)]- (6-10), or 4-[3-(1-methoxycarbonyl-1,2-(and 1,6-)dihydropyridyl)]- (16-20), moieties were synthesized by reaction of the 4-(4-pyridyl)- (1-5), or 4-(3-pyridyl)- (11-15), precursors with methyl chloroformate in the presence of sodium borohydride. In vitro calcium channel (CC) antagonist activities were determined using a muscarinic-receptor-mediated Ca 2+ -dependent contraction of guinea pig ileal longitudinal smooth muscle (GPILSM) assay. Elaboration of the 4-pyridyl (1-5), or 3-pyridyl (11-16), substituent to the corresponding 1,2-(1,6)dihydropyridyl moiety abolished the CC agonist effect produced by 1-5 and 11-15 on GPILSM. In addition, all alkyl esters possessing a 4-[4- or 3-(1-methoxycarbonyl-1,2--(1,6-)dihydropyridyl)]- moiety (6-10, 17, 19-20) were inactive CC antagonists on GPILSM with the exception of the ethyl (16) and isobutyl (18) ester derivatives, which exhibited weak CC antagonist activity. All of the 4-[4- or 3-(1-methoxycarbonyl-1,2-(1,6-)dihydropyridyl)] compounds evaluated (6-10, 16-19) exhibited in vitro CC agonist (positive inotropes) activity on guinea pig left atria (GPLA). In general, the 4-[3-(1-methoxycarbonyl-1,2-(and 1,6)dihydropyridyl)] compounds (16-19) were more potent CC agonists (EC 50 = 2.91 × 10 -6 to 7.51 x 10 -6 M range) than the corresponding 4-[4-(1-methoxycarbonyl-1,2-dihydropyridyl)] compounds (6-9, EC 50 = 1.42 x 10 -5 to 2.65 x 10 -5 M range). These 4-[4- or 3-(1-methoxycarbonyl-1,2-(1,6)dihydropyridyl)] compounds, which exhibit cardiospecific CC agonist activity (6-10, 17, 19) will serve as valuable model compounds to study the structure-function relationships of CC modulation, and provide a new drug design concept directed toward the treatment of congestive heart failure.

Novel Hantzsch 1,4-dihydropyridines to study the structure-function relationships of calcium channels and photoinduced relaxation

A group of methyl 1,4-dihydro-2,6-dimethyl-4-(2-, 3- or 4-NHOH; 3- or 4-N=O)-phenyl-5-pyridinecarboxylates possessing a C-3 CO2Me or NO2 substituent [compounds 5–8, 10–12, below] were synthesized by reduction of the C-4 nitrophenyl precursors [1–4] to the corresponding phenylhydroxylamine [5–8] derivatives using 5% rhodium-on-charcoal with hydrazine hydrate as the hydrogen donor, followed by re-oxidation of the phenylhydroxylamine product [6–8] to the corresponding nitrosophenyl [10–12] derivative using pyridinium chlorochromate. A series of 1,4-dihydro-2,6-dimethyl-4-(2-trifluoromethylphenyl)pyridines [26–34] possessing CO2Me, COMe, CONH2, P(=O)OEt2, CN, NO2 C-3/C-5 substituents were synthesized using a modified Hantzsch reaction involving the condensation of 2-trifluoromethylbenzaldehyde [17] with an aminocrotonate [18–20] and a ketone [21–25] derivative. In vitro calcium channel (CC) activities were determined using a muscarinic-receptor-mediated Ca+2-dependent contraction of guinea pig ileal longitudinal smooth muscle assay. This class of compounds [5–8, 10–12, 26–34] exhibited weak CC antagonist activity [10–4 to 10–7 M range] relative to the reference drug nifedipine [IC50 = 1.4 × 10–8 M]. Structure–activity relationships [SARs] acquired were in agreement with known SARs where the relative potency order for C-4 phenyl substituents is ortho and meta > para. A C-3 nitro substituent decreased CC antagonist activity. Compounds 29–34 possessing C-3 CN or NO2, and a C-5 CO2Me, NO2, CONH2, COMe, or P(=O)OEt2, substituents exhibited weak CC antagonist activity in the 10–4 to 10–5 M range. Although this group of highly functionalized 1,4-dihydropyridines are not useful CC antagonists, they will serve as valuable model compounds to study the structure–function relationships of CC modulation. Drug Dev. Res. 42:120–130, 1997. © 1997 Wiley-Liss, Inc.

Effects of various selective phosphodiesterase inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle.

The effects of various selective phosphodiesterase (PDE) inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle were investigated. 1) 3-Isobutyl-1-methyl xanthine (IBMX) or zaprinast markedly inhibited the high K(+)- or carbachol (CCh)-induced contraction and increased cGMP content of the muscle strip in a concentration-dependent manner. However, these agents only slightly increased the cAMP content. Milrinone or Ro20-1724 also slightly inhibited the high K(+)- or CCh-induced contraction and increased the cAMP content, but did not increase cGMP. 2) In a fura2-loaded muscle, IBMX or zaprinast inhibited both contractions and the increase in intracellular Ca2+ ([Ca2+]i) level induced by high K+ or CCh, although the inhibitory effect on the [Ca2+]i level was smaller than that on muscle tension. 3) In alpha-toxin-permeabilized muscles, cGMP, IBMX or zaprinast significantly inhibited the Ca(2+)-induced contraction. These results suggest that IBMX and zaprinast inhibit muscle contraction in the ileal longitudinal smooth muscles mainly through an increase in cGMP and the inhibitory mechanism of IBMX or zaprinast is involved in the decreases in the [Ca2+]i level and sensitivity of contractile elements to Ca2+.

Effects of Various Selective Phosphodiesterase Inhibitors on Muscle Contractility in Guinea Pig Ileal Longitudinal Smooth Muscle

Effects of Various Selective Phosphodiesterase Inhibitors on Muscle Contractility in Guinea Pig Ileal Longitudinal Smooth Muscle

P-236 - Effects of selective phosphodiesterase inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle.

P-236 - Effects of selective phosphodiesterase inhibitors on muscle contractility in guinea pig ileal longitudinal smooth muscle.

The Inhibitory Effect of Li+ on Contractile Elements of Intestinal Smooth Muscle

The mechanism of the inhibitory effect of Li+ on contraction was examined in guinea pig ileal longitudinal smooth muscle. Li+-substitution (68.4 mM) reversed contractions induced by high K+ (45.4 mM), carbachol (1 ¿/M) and histamine (1 μM) without changing the cytosolic Ca2+ level. Li+ also had no effect on the increase in 45Ca2+ uptake stimulated by high K+. High K+ transiently increased myosin light chain (MLC) phosphorylation, reaching a peak at 6-9 sec. Li+-substitution inhibited the high K+-induced MLC phosphorylation. In permeabilized ileal strips, contraction induced by 1 μM Ca2+ was inhibited by 10 mM Li+. The inhibitory effect was antagonized by increasing the concentration of Ca2+ or calmodulin. In the permeabilized muscle in which MLC was previously thiophosphorylated with 1 mM ATPfS and 3 μM Ca2+, ATP induced contraction in Ca2+ free buffer. Li+ added during this contraction did not show an inhibitory effect. In contrast, when 30 mM Li+ was added during the thiophosphorylation, the contraction induced by the subsequent addition of ATP was inhibited. Li+ (30 mM) changed neither the rate of relaxation induced by removing external Ca2+ in permeabilized muscle nor the rate of dephosphorylation of MLC induced by crude phosphatase extracted from the ileum. Li+ (15 mM), on the other hand, inhibited the rate of phosphorylation of MLC caused by crude MLC kinase extracted from the ileum. Li+ did not inhibit the calmodulin activity as measured with the (Ca2+ + Mg2+)-ATPase activity of the erythrocyte membrane. These results suggest that the inhibitory effect of Li+ on contractions is attributable to the inhibition of MLC kinase in guinea pig ileum.

Decrease in Muscle Tension and Reduced Pyridine Nucleotides of the Guinea Pig Ileal Longitudinal Smooth Muscle in High K+,Na+-Deficient Solution.

In the present experiment, we studied the inhibitory mechanism of Na+ depletion on high K(+)-induced contraction by simultaneously measuring reduced pyridine nucleotides (PNred) or oxidized flavoproteins (FPox) fluorescence and contractile tension of the guinea pig ileal longitudinal muscle. Tension, PNred and FPox were all reversibly increased by the addition of hyperosmotic 65 mM KCl (H-65K+). A high K+, Na(+)-deficient (Iso-154K+) solution induced a contraction followed by a gradual relaxation and gradually decreased PNred fluorescence. A hyperosmotic addition of NaCl to the Iso-154K+ solution prevented the decreases in tension and PNred fluorescence. Addition of pyruvate or oxaloacetate restored the decrease in Iso-154K(+)-induced contraction, but not the decrease in PNred fluorescence. In contrast to the PNred fluorescence, an application of the Iso-154K+ solution increased the FPox fluorescence which was not significantly changed by an addition of NaCl, pyruvate or oxaloacetate. These results suggest that the inhibitory mechanism of Na+ depletion on the Iso-154K(+)-induced contraction is an inhibition of glucose utilization.

Decrease in Muscle Tension and Reduced Pyridine Nucleotides of the Guinea Pig Ileal Longitudinal Smooth Muscle in High K +, Na +-Deficient Solution

Decrease in Muscle Tension and Reduced Pyridine Nucleotides of the Guinea Pig Ileal Longitudinal Smooth Muscle in High K +, Na +-Deficient Solution

Irreversible modification of the voltage-sensitive calcium channel by N-ethoxycarbonyl-2-ethyoxy-1,2-dihydroquinoline (EEDQ)

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inhibited, in vitro, the specific binding of three structurally distinct L-type Ca 2+ channel ligands, (+)[ 3H]PN 200 110, [ 3H]desmethoxyverapamil and [ 3H] cis-diltiazem to guinea pig ileal longitudinal smooth muscle. Maximum tension responses to Ca 2+ in a K +-depolarized functional smooth muscle preparation were reduced in a concentration-dependent manner following pretreatment with EEDQ and washout. Microsomal membranes prepared from smooth muscle pretreated with EEDQ followed by extensive washout showed a significant reduction in the amount of (+)[ 3H]PN 200 110 bound without change of ligand affinity. Similar results were obtained in cardiac ventricle microsomes. Preincubation with verapamil (1 × 10 −5 M) largely prevented this reduction in [ 3H]PN 200 110 binding sites by EEDQ. 45Ca 2+ uptake in cortical synaptosomes during 1-sec depolarization following 68.5 mM K + was also inhibited by EEDQ. Specific binding of [ 125I]ω-conotoxin GVIA to rat cerebral cortex membranes was inhibited by EEDQ, also in an apparently irreversible manner as seen by the marked reduction in binding site density with no significant change in the K D value. These observations indicate that EEDQ blocks Ca 2+ channel function and reduces irreversibly both 1,4-dihydropyridine and ω-conotoxin GVIA binding sites.

Synthesis and calcium channel antagonist activity of 3-arylmethyl 5-isopropyl 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates.

Unsymmetrical aryl(heteroaryl)methyl isopropyl ester analogues of nifedipine, in which the 2-nitrophenyl group at C-4 is replaced by a 2- or 3-pyridyl substituent, were synthesized and evaluated as calcium-channel antagonists using guinea pig ileal longitudinal smooth muscle. The point of attachment of the C-4 pyridyl substituent was a determinant of activity where the relative potency order was 2-pyridyl greater than 3-pyridyl. Within the C-4 2-pyridyl series of compounds, and electronegative substituent such as a trifluoromethyl or bromo at the 4 position of the benzyl ester substituent or a nitrogen atom at the 1 position of a 4-pyridylmethyl ester substituent, enhanced activity relative to the unsubstituted benzyl ester analogue. In contrast, in the C-4 3-pyridyl class of compounds, a variety of aryl(heteroaryl)methyl ester substituents did not alter potency to any significant extent. A number of compounds in the C-4 2-pyridyl series possessing 4-pyridylmethyl, 4-trifluoromethylbenzyl, 4-bromobenzyl, and 3-pyridylmethyl ester substituents were approximately equipotent to nifedipine. The aryl(heteroaryl)methyl ester and C-4 2-pyridyl substituents therefore appear to provide important interdependent contributions to calcium-channel antagonist activity.

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle.

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.

In vitro and in vivo characterization of 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyano)phenyl-1,4- dihydropy ridine as a Ca2+ channel antagonist.

The actions of 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyano)phenyl-1,4- dihydropyridine (o-NCS-DHP), a nifedipine analog bearing a reactive group, have been characterized in vitro by pharmacological and radioligand binding techniques in a number of smooth muscles and in vivo by blood pressure and radioligand binding. o-NCS-DHP exhibits persistent, but slowly reversible, antagonism in guinea pig ileal longitudinal smooth muscle, guinea pig bladder, taenia coli, rat portal vein, and rat tail artery to receptor responses (muscarinic and alpha-adrenoceptor) and K+ depolarization initiated responses. Duration of response was significantly longer than that of equivalent concentrations of nifedipine. In many tissues a component of antagonism produced by o-NCS-DHP was not reversed by repeated washing over the duration of the experiment (up to 2 or 7 h). A comparison of the actions of o-NCS-DHP and its isomers m-NCS-DHP and p-NCS-DHP revealed the former to be significantly longer lasting in rat tail artery against K+ depolarization induced responses. A similar profile was exhibited when the Ca2+ channel activator Bay K 8644 was employed as the stimulant, but the antagonism produced by all three compounds was fully reversed with sufficiently prolonged washing. In vivo administration of o-NCS-DHP (5-25 mg/kg) produced a persistent reduction of [3H]nitrendipine binding in rat brain, gut, and heart characterized as Bmax, but not KD, changes. No effects on [3H]dihydroalprenolol or [3H]quinuclidinyl benzilate binding were detected. Binding site recoveries were characterized by t1/2 values of 35-50 h, and these were significantly prolonged to 91-107 h in animals treated with cycloheximide. Recovery of [3H]nitrendipine binding sites correlated with blood pressure restoration in spontaneously hypertensive rats. These data suggest that o-NCS-DHP possesses both reversible and irreversible actions. The reversible actions are unusually persistent compared with nifedipine and other 1,4-dihydropyridine analogs. This persistent, but reversible component, may be accompanied by an irreversible action particularly at the higher concentrations employed in the in vivo experiments.

Synthesis and calcium channel antagonist activity of dialkyl hexahydro-1,2′,6′-trimethyl[bipyridine]-3′,5′-dicarboxylates

Reaction of dialkyl 1′,4′-dihydro-2′,6′-dimethyl[bipyridine]-3′,5′-dicarboxylates 2 with methyl iodide yielded the corresponding 4′-(1-methylpyridinium) iodide salts 3 in quantitative yield. The sodium borohydride reduction of 3 in aqueous ethanol gave the corresponding dialkyl hexahydro-1,2′,6′-trimethyl[bipyridine]-3′,5′-dicarboxylate analogs 4–5. The calcium channel antagonist activities for 4–5 were determined using the muscarinic receptor-mediated Ca 2+-dependent contraction of guinea pig ileal longitudinal smooth muscle. The relative activities for the 3′,5′-diethyl series 5 was 3-tetrahydropyridinyl 5b > 4-tetrahydropyridinyl 5c > 2-tetrahydropyridinyl 5a. Increasing the size of the 3′,5′-alkyl substituents enhanced activity. An approximate 1:1 correlation between the IC 50 value for the inhibition of [ 3H]nitrendipine binding and inhibition of the tonic component of the muscarinic-induced contractile response was observed for 4a and 5b. NMR studies suggest that the 4′-[2-(1-methyl-1,2,3,6-tetrahydropyridinyl)] ring systems of 4a and 5a are anti-periplanar to the 1,4-dihydropyridine ring system.

Interaction of phenoxybenzamine with muscarinic receptors and calcium channels

Phenoxybenzamine (POB, 10 −6–10 −4 M) inhibited the responses of guinea pig ileal longitudinal smooth muscle to both muscarinic agonists and K + -depolarization but was more effective against the agonist-induced responses. POB inhibited binding of both the muscarinic antagonist [ 3H]-quinuclidinyl benzilate (QNB) and the Ca 2+ channel antagonist [ 3H]nitrendipine and was, paralleling its effects on mechanical responses, more effective against [ 3H]QNB binding. POB reduced specific [ 3H]QNB binding by a reduction in B max with no change in K D , but inhibited [ 3H]nitrendipine binding by reducing k D with no effect on B max. It is suggested that the activity of POB against Ca 2+ channels may underlie the ability of POB, and other 2-halogenoethylamines, to inhibit a wide variety of apparently discrete pharmacological events.