M. Hawthorn

The actions of peppermint oil and menthol on calcium channel dependent processes in intestinal, neuronal and cardiac preparations

The activities of menthol and peppermint oil were determined in guinea‐pig ileal smooth muscle, in rat and guinea‐pig atrial and papillary muscle, in rat brain synaptosomes and in chick retinal neurones by pharmacological 45Ca2+ uptake and radioligand binding assays. Menthol is a major constituent of peppermint oil and is approximately twice as potent as peppermint oil as an inhibitor of K+ depolarization‐induced and electrically stimulated responses in ileum and electrically stimulated atrial and papillary muscles. IC50 values in the ileal preparation ranged from 7.7 to 28.1 μg ml−1 and in the cardiac preparations from 10.1 to 68.5 μg ml−1. Similar potencies were demonstrated against K+ depolarization‐induced 45Ca2+ uptake in synaptosomes and against K+ depolarization and Bay K 8644‐induced uptake in chick retinal neurons. IC50 values for menthol inhibition of K+ and Bay K 8644 responses in the retinal neurons were 1.1 × 10−4 M (17.2 μg ml−1) and 1.75 × 10−4 M (26.6 μg ml−I), respectively, and for peppermint oil were 20.3 and 41.7 μg ml−1 respectively. Both menthol and peppermint oil inhibited specific [3H]nitrendipine and [3H]PN 200–110 binding to smooth and cardiac muscle and neuronal preparations with potencies comparable to, but slightly lower than, those measured in the pharmacological and 45Ca2+ uptake experiments. Binding of menthol and peppermint oil, studied at 78 μg ml−1, was competitive against [3H]nitrendipine in both smooth muscle and synaptosome preparations. The data indicate that both menthol and peppermint oil exert Ca2+ channel blocking properties which may underlie their use in irritable bowel syndrome. Ca2+ channel antagonism may not be the only pharmacological effect of menthol and peppermint oil contributing to intestinal smooth muscle relaxation.

Effect of an homologous series of aliphatic alcohols on neuronal and smooth muscle voltage-dependent Ca2+ channels

The acute inhibitory actions of alcohol on K(+)-stimulated 45Ca2+ uptake into synaptosomes shows regional variation in sensitivity throughout the brain, suggesting the possibility of a selective action on a specific Ca2+ channel subtype. This was examined by comparing the effects of a homologous series of aliphatic alcohols on synaptosomal Ca2+ channels with their actions on K(+)-stimulated Ca2+ channels in guinea-pig intestinal longitudinal muscle, which have been demonstrated to be of the L-type. K(+)-stimulated contraction of and [3H]nitrendipine binding to smooth muscle were both inhibited by the alcohols at similar concentrations, with the potency increasing with chain length. In synaptosomes, however, K(+)-stimulated 45Ca2+ uptake was 5-30 times more sensitive to the inhibitory actions of alcohol than were [3H]nitrendipine and [125I]omega-conotoxin binding. These observations suggest that K(+)-stimulated 45Ca2+ uptake is mediated by a non-L non-N type channel which is more sensitive to the acute effects of alcohols. This is supported by the observation that K(+)-stimulated 45Ca2+ uptake which is insensitive to L- and N-channel antagonists was inhibited by funnel web spider venom.

Potential-Dependent Interactions of Nitrendipine and Related 1,4-Dihydropyridines in Functional: Smooth Muscle Preparations

Interaction of nitrendipine and other Ca2+ channel antagonists including nifedipine, diltiazem, and D600 with intestinal smooth muscle was shown to depend on membrane potential. In the absence of extracellular Ca2+, guinea pig ileal longitudinal muscle does not contract and can be incubated at various K+ concentrations in the presence or absence of antagonist. Preincubation with elevated K+ prior to admission of Ca2+ and challenge with K+ to a total of 100 mM increased the activity of the antagonist. The IC50 for nitrendipine incubated in the presence of 5 mM K+ was 5.36 X 10(-9) M and in the presence of 40 mM K+ was 0.53 X 10(-9) M. Other 1,4-dihydropyridines showed similar potency shifts.

Iminodipropionitrile-Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists

Administration of 3, 3′‐iminodipropionitrile (IDPN) (1g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN‐induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200–110 binding sites (Bmax) in whole brains of IDPN‐treated mice. Similarly, the K+‐depolarization‐dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 ± 7 fmol/mg to 237 ± 31 fmol/mg in control and treated groups, respectively) of PN 200–110 binding to the striatum without change of KD value (38 ± 4.7 pM versus 33 ± 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 ± 10.1 pM to 45 ± 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 ± 51 fmol/mg versus 485 ± 41 fmol/mg) of PN 200–110 binding to the cortex. IDPN did not alter ω‐conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment are inhibited by L‐type calcium channel antagonists and this may be associated with the observed increase in striatal L‐type calcium channels.

Thermodynamic and kinetic aspects of agonist and antagonist binding to 1,4-dihydropyridine receptors

The kinetic and equilibrium binding properties of the 1,4-dihydropyridine activator [3H](-)-S-Bay K 8644 and the antagonist [3H](+)-PN 200-110 were determined in rat heart membrane particulate preparations at temperatures between 4 and 37 degrees C. The binding of [3H](-)-S-Bay K 8644 was temperature-dependent with a single binding site with KD = 3.57 nM and Bmax = 330 fmol/mg.protein at 25 degrees C. The association and dissociation rate constants were 3.4 x 10(7) min-1 M-1 and 0.095 min-1 respectively at 25 degrees C and decreased slightly at lower temperatures. In contrast, [3H](+)-PN 200-110 bound to high (KD(H) = 0.032 nM, Bmax(H) = 316 fmol/mg.protein) and low affinity sites (KD(L) = 27.6 nM and Bmax(L) = 6432 fmol/mg.protein) at 25 degrees C in rat heart preparation. A similar two-site binding of [3H](+)-PN 200-110 was found in rat brain preparation, but only a single binding site was detected in rat skeletal muscle. Binding of [3H](+)-PN 200-110 to the high and low affinity sites in cardiac membranes was sensitive and insensitive respectively to temperature. Association and dissociation rates of [3H](+)-PN 200-110 at the high affinity binding sites were best fitted as mono-exponential functions. Association and dissociation rates of [3H](+)-PN 200-110 were 3.94 x 10(8) min-1 M-1 and 7.86 x 10(-3) min-1 at 25 degrees C. The association rate varied only slightly (3-fold), but the rate of dissociation decreased significantly (200-fold) with temperature from 37 to 4 degrees C. Thermodynamic analysis of equilibrium binding showed that the binding of activator was enthalpy driven, whereas the binding of antagonist to the high affinity site was both entropy- and enthalpy-driven and to the low affinity site was totally entropy-driven.

Chronic ethanol exposure potentiates muscarinic receptor and α2-adrenoceptor-mediated inhibition of cAMP accumulation in PC 12 cells

The effects of ethanol on receptor-mediated inhibition of cAMP production were investigated in PC 12 cells. The in vitro addition of ethanol enhanced N-ethylcarboxyadenosine (NECA)-stimulated cAMP production without altering the inhibitory action of carbachol or epinephrine. A 4 day exposure of PC 12 cells to ethanol decreased the stimulation of cAMP production by NECA, but increased the inhibition of NECA-stimulated cAMP production by carbachol and epinephrine.