Changes in Ca(2+)/calmodulin-dependent protein kinase II activity and its relation to performance in passive avoidance response and long-term potentiation formation in mice prenatally exposed to diethylstilbestrol.

We investigated the effects of prenatal exposure to diethylstilbestrol (DES), an endocrine disrupter on learning behavior and synaptic functions. Specifically, we determined the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and related kinases that play an essential role in long-term potentiation (LTP) in the hippocampus in mice that were prenatally exposed to DES. Treatment with DES resulted in increased CaMKII autophosphorylation and Ca(2+)-independent activity in the hippocampus and cortex of male mice. Impaired passive avoidance correlated with this increased CaMKII autophosphorylation, as did the enhanced early phase of LTP (E-LTP) in hippocampus. These data suggest that prenatal exposure to DES induces deficits in passive avoidance responses as a result of increased CaMKII activity and hippocampal LTP.

Pharmacological Properties of Propiverine Contribute to Improving Lower Urinary Tract Dysfunctions in Rats with Spinal Cord Injuries.

Patients with spinal cord injury (SCI) usually develop lower urinary tract dysfunctions, including detrusor overactivity which is also known to be a risk factor for upper urinary tract dysfunction. Antimuscarinic agents, such as propiverine, have been used clinically for the treatment of detrusor overactivity. Also, propiverine has been known to possess antagonistic activity against L-type Ca2+ channels and transient receptor potential vanilloid subtype 1 (TRPV1), in addition to activity against muscarinic receptors. These mechanisms of action may contribute to improving detrusor overactivity in SCI. We therefore investigated the effects of antagonists of these mechanisms on non-voiding contraction (NVC) in SCI rats that are similar to clinical cases of detrusor overactivity, and considered whether these action mechanisms contribute to the incidence of NVC in SCI. Cystometry was performed in rats 4 weeks after spinal transection. Urinary functions were evaluated before and after intravenous administration of propiverine and specific antagonists for muscarinic receptors (atropine), L-type Ca2+ channels (verapamil), and TRPV1 (capsazepine). Propiverine markedly decreased the amplitude pressure of NVC in SCI rats, which was partially inhibited by atropine. Verapamil also suppressed the amplitude pressure of NVC to the same degree as propiverine. NVC disappeared almost completely after C-fiber desensitization, although capsazepine exerted no evident effects. These findings suggest that muscarinic receptors, L-type Ca2+ channels, and C-fiber afferent nerves contribute to the incidence of detrusor overactivity in SCI, and a drug that has multiple antagonistic effects, such as propiverine, is very effective for the treatment of lower urinary tract dysfunctions in SCI.

A family with Liddle's syndrome caused by a new missense mutation in the beta subunit of the epithelial sodium channel.

Liddle's syndrome is an autosomal dominant form of salt sensitive hypertension caused by mutations in the beta or gamma subunit of the epithelial sodium channel. Systematic mutagenesis studies revealed that a conserved PPPXY sequence (PY motif) of the C-terminus of the alpha, beta, or gamma subunits might be involved in the regulation of the channel activity. However, only two missense mutations in the PY motif of the beta subunit have been reported to cause Liddle's syndrome. We sequenced the C-termini of the beta and gamma subunits of the epithelial sodium channel in a Japanese family clinically diagnosed as having Liddle's syndrome and found a new missense mutation in the PY motif of the beta subunit, P615S. Expression studies with P615S mutant in Xenopus oocytes resulted in an about 3-fold increase in the amiloride-sensitive sodium current compared to the wild type (p = 0.001). These findings provide further clinical evidence for the hypothesis that a conserved PY motif may be critically important for the regulation of the epithelial sodium channel.

Inhibition of the 5-HT(1A) receptor-mediated inwardly rectifying K(+) current by dextromethorphan in rat dorsal raphe neurones.

The effect of dextromethorphan (DM) on the inwardly rectifying K(+) currents mediated by 5-HT(1A) receptors in acutely dissociated dorsal raphe (DR) neurones of rats was studied using nystatin-perforated patch and conventional whole-cell patch recording configurations under voltage-clamp conditions. DM rapidly and reversibly inhibited the K(+) currents induced by 10(-7) M 5-HT in a concentration-dependent manner with a half-maximum inhibitory concentration of 1.43 x 10(-5) M. The inhibitory effect of DM was neither voltage- nor use-dependent. DM caused a suppression of the maximum response of the 5-HT concentration-response curve, thus suggesting a non-competitive type of inhibition. In neurones perfused intracellularly with a pipette-solution containing the nonhydrolyzable GTP analog GTPgammaS, 5-HT activated K(+) currents in an irreversible manner. DM suppressed the current irreversibly activated by intracellular GTPgammaS even in the absence of the agonist. DM also inhibited the inwardly rectifying K(+) currents regulated by alpha(2)-adrenoceptors in freshly isolated rat locus coeruleus neurones. These results suggest that DM may inhibit the G-protein coupled inwardly rectifying K(+) channels, but not the neurotransmitter receptors, in the central nervous system.

Piperidine: a microelectrophoretic study in the mammalian brain.

Using unit recording and electrophoretic techniques, the action of piperidine on unit activity of the brain of the rat was studied. Piperidine excited 31%, and inhibited 4% of cortical cells tested. In the hippocampus and caudate nucleus, piperidine excited larger proportions of the cells tested. The actions of piperidine were blocked by tetraethylammonium but not by scopolamine.

Pipecolic acid enhancement of GABA response in single neurons of rat brain.

Using unit recording and microelectrophoresis, influence of pipecolic acid (PA), a major metabolite of lysine in the brain, on GABA and glycine responses was studied in the cerebral cortical and hippocampal pyramidal neurons of rats. With small currents, PA had no effect on the single neuron activities but enhanced GABA response without affecting glycine response. The finding provides a new evidence that PA may have a connection with central GABA system.

Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents.

The action of bradykinin on neurons acutely isolated from airway parasympathetic ganglia of rats and its mechanism were investigated using the nystatin-perforated patch-clamp recording technique. Under current clamp conditions, an application of 0.1 microM bradykinin onto rat airway ganglion neurons induced a depolarization which was accompanied by the action potential firing. Bradykinin elicited inward currents with decreasing the membrane conductance when a ganglion neuron was held at a holding potential of -40 mV. The half-maximum effective concentration was 8.9 nM. The bradykinin response was mimicked by a B(2) receptor agonist, [Hyp(3)]-bradykinin, and was inhibited by HOE-140, a B(2) antagonist, suggesting the contribution of B(2) receptors. The bradykinin-induced inward current reversed at the K(+) equilibrium potential, which shifted 56.5 mV with a 10-fold change in extracellular K(+) concentration. The application of 10(-3) M Ba(2+) induced the inward current, and bradykinin failed to evoke a further inward current in the presence of Ba(2+). Bradykinin also reduced the amplitude of M-current deactivation induced by a hyperpolarizing step from a holding potential of -25 mV to -50 mV with a half-maximum effective concentration of 16 nM. Pretreatment with pertussis toxin had no effect on the bradykinin-induced inhibition of the M-current. From these results we suggest that bradykinin may be able to depolarize the airway parasympathetic ganglion neurons of rats associated with an inhibition of M-type K(+) channels through the B(2) type of bradykinin receptors.

Inhibition of glycine currents by dextromethorphan in neurones dissociated from the guinea-pig nucleus tractus solitarii.

1. The effect of dextromethorphan (DM) on the current induced by glycine in acutely dissociated nucleus tractus solitarii (NTS) neurones of guinea-pigs was studied by use of the whole-cell patch clamp technique. The effect of DM on gamma-aminobutyric acid (GABA)-induced currents (IGABA) was also examined. 2. DM inhibited 30 microM glycine-induced current (IGly), without affecting the current caused by 30 microM GABA. The action of DM was concentration-dependent, with a maximum effect at 100 microM, and reversible. The half-maximum inhibitory concentration (IC50) of DM was 3.3 microM, about 85 times higher than that of strychnine. 3. DM 3 microM shifted the concentration-response curve for glycine to the right without affecting the maximum value. DM 10 microM shifted the curve even more to the right, although it was not a parallel shift. Strychnine at a concentration of 0.1 microM shifted the curve for glycine in a nearly parallel fashion. 4. The effect of 10 microM DM was slightly weak voltage-dependency, but the lower concentration of DM, 3 microM, inhibited IGly equally at -50 mV and +50 mV. The effect of 3 microM DM on IGly showed no use-dependence. Blockade by strychnine 0.1 microM showed no voltage- or use-dependence. 5. The results indicate that DM inhibits IGly in single neurones of NTS, and further suggest that DM at a low concentration may act on the glycine receptor-ionophore complex, but not on the Cl channel of the complex. However, a relatively high concentration of DM may at least partly affect the Cl- channel of the complex.

Activation of potassium conductance by ophiopogonin-D in acutely dissociated rat paratracheal neurones.

1. The effect of ophiopogonin-D (OP-D), a steroidal glycoside and an active component of Bakumondo-to, a Chinese herbal antitussive, on neurones acutely dissociated from paratracheal ganglia of 2-week-old Wistar rats was investigated using the nystatin-perforated patch recording configuration. 2. Under current-clamp conditions, OP-D (10 microM) hyperpolarized the paratracheal neurones from a resting membrane potential of -65.7 to -73.5 mV. 3. At the concentration of 1 microM and above, OP-D concentration-dependently activated an outward current accompanied by an increase in the membrane conductance under voltage-clamp conditions at a holding potential of -40 mV. 4. The reversal potential of the OP-D-induced current (I(OP-D)) was -79.4 mV, which is close to the K(+) equilibrium potential of -86.4 mV. The changes in the reversal potential for a 10 fold change in extracellular K(+) concentration was 53.1 mV, indicating that the current was carried by K(+). 5. The I(OP-D) was blocked by an extracellular application of 1 mM Ba2+ by 59.0%, but other K(+) channel blockers, including 4-aminopyridine (3 mM), apamin (1 microM), charybdotoxin (0.3 microM), glibenclamide (1 microM), tolbutamide (0.3 mM) and tetraethylammonium (10 mM), did not inhibit the I(OP-D). 6. OP-D also inhibited the ACh- and bradykinin-induced depolarizing responses which were accompanied with firing of action potentials. 7. The results suggest that OP-D may be of benefit in reducing the excitability of airway parasympathetic ganglion neurones and consequently cholinergic control of airway function and further, that the hyperpolarizing effect of OP-D on paratracheal neurones via an activation of K(+) channels might explain a part of mechanisms of the antitussive action of the agent.

Piperidine discriminates between the transient and the persistent components of the ACh-induced chloride current in Aplysia neurons.

ACh-induced Cl- -current (ICl) is well known to desensitize with two components: an initial fast phase followed by a second, more slowly developing phase. In the present study, the influence of piperidine, a normal constituent in vertebrates and invertebrates, on ACh-induced ICl in isolated neurons of Aplysia was investigated by using the concentration clamp in combination with the voltage clamp technique. Pretreatment with piperidine in doses greater than 2 X 10(-4)M depressed the transient ACh-induced ICl but had little effect on the persistent ICl. Kinetic study of the desensitization phase of ACh-induced ICl showed that the slow time constant of the desensitization phase of ACh-induced ICl was not altered by pretreatment with piperidine. The present results indicate that piperidine can discriminate between the fast transient and slow persistent components of ACh-induced ICl in Aplysia neurons, and also suggest that two components of the desensitization phase of ACh-induced ICl function in an independent manner.

Influence of cerebral cortex stimulation upon cough-like spasmodic expiratory response (SER) and cough in the cat.

In 27 pentobarbitalized cats, the influence of electrical stimulation of the cerebral cortex upon the spasmodic expiratory response (SER) was studied and compared with cortical influences on coughing induced by stimulation of the superior laryngeal nerve (sup. laryngeal N.). This cortical influence was evoked by electrical stimulation of the cortical nucleus of amygdala (Aco), and was very similar to coughing accompanying changes in emotional behavior and was depressed more effectively by psychotropics than by centrally acting antitussives like codeine. When anterior cingulate gyrus (ant. cingulate G.) or orbital gyrus (orbital G.) were stimulated simultaneously with Aco or sup. laryngeal N., weak stimulation was sufficient to inhibit SER, while stronger stimuli were needed for the suppression of cough. If the same cortical regions were stimulated after initiation of SER or cough, SER was markedly suppressed but cough little affected. Production of SER was facilitated by simultaneous stimulation of the piriform lobe (piriform L.) or olfactory tract (olfactory T.), whereas cough production was facilitated by simultaneous stimulation of the suprasylvian gyrus. These results suggest that SER and coughing are differently controlled by the cerebral cortex, and that SER is modulated by the limbic cortex, in particular, by ant. cingulate G., orbital G. and piriform L. The mechanism of modulation for SER is discussed.

Preferential inhibition of L- and N-type calcium channels in the rat hippocampal neurons by cilnidipine.

The effect of a dihydropyridine Ca2+ antagonist, cilnidipine, on voltage-dependent Ca2+ channels was studied in acutely dissociated rat CA1 pyramidal neurons using the nystatin-perforated patch recording configuration under voltage-clamp conditions. Cilnidipine had no effect on low-voltage-activated (LVA) Ca2+ channels at the low concentrations under 10(-6) M. On the other hand, cilnidipine inhibited the high-voltage-activated (HVA) Ca2+ current (I(Ca)) in a concentration-dependent manner and the inhibition curve showed a step-wise pattern; cilnidipine selectively reduced only L-type HVA I(Ca) at the low concentrations under 10(-7) and 10(-6) M cilnidipine blocked not only L- but also N-type HVA I(Ca). At the high concentration over 10(-6) M cilnidipine non-selectively blocked the T-type LVA and P/Q- and R-type HVA Ca2+ channels. This is the first report that cilnidipine at lower concentration of 10(-6) M blocks both L-and N-type HVA I(Ca) in the hippocampal neurons.

Seasonal and activity-dependent variations of piperidine levels in the amphibian brain.

Piperidine is one of biogenic amines possessing nicotine-like synaptotropic actions on the nervous systems. Since piperidine produces multiplex physiological actions, a role for the amine as a modulator in neuroendocrine as well as neuronal functions has been supposed. In the present study, piperidine levels in the amphobian brains during activity and hibernation were examined by use of a mass fragmentographic technique and it was found that the brain piperidine concentrations significantly increased in the cold season especially during hibernation. The significance of the findings is discussed with respect to the hypnogenic effect of piperidine.

Electrophoretic study of pipecolic acid, a biogenic imino acid, in the mammalian brain.

Pipecolic acid (PA), one of the imino acids, is a normal constituent in the mammalian brain. It is said that PA is a major intermediate of lysine metabolism in the rat brain. Biochemical studies have suggested that PA may be involved in the regulation of synaptic mechanism in the CNS. Moreover, the pathophysiological significance of PA has been also suggested by some investigators. However, there has so far been no good evidence based on the comprehensive electrophysiological experiments. Using unit recording and microelectrophoretic technique, the action of PA on single neuron activities in the rat brain was examined. PA depressed the firing of 88 out of 115 cortical neurons tested. Only 2 were excited and 25 remained unaffected. All the identified hippocampal pyramidal neurons examined were uniformly inhibited. It has been reported that PA inhibits the uptake of GABA into the brain slices and enhances the release of GABA from the slices. Thus, it is likely that the inhibitory response due to PA may have some connections with GABAergic transmission. On the other hand, it remains to be clarified whether the specific PA sensitive receptors exist in the brain. Our findings provide a clue to the elucidation of the presumed synaptic involvement of PA in the CNS.

Pipecolic acid: a new type of alpha-amino acid possessing bicuculline-sensitive action in the mammalian brain.

Using unit recording and electrophoretic techniques, pharmacological properties of pipecolic acid (PA) were studied in the brain neurons of rats. PA response was blocked by bicuculline more effectively than GABA response but not blocked by strychnine. Stereochemical findings obtained using the HGS-model demonstrated that PA structure is almost the same as a part of bicuculline structure. The present results suggest that PA might be a new type of substance possessing bicuculline-sensitive action. The site of the action of PA was also discussed.

Differential effect of codeine on coughs caused by mechanical stimulation of two different sites in the airway of guinea pigs.

We studied the difference in the effects of codeine on coughs caused by mechanical stimulation to the larynx and to the bifurcation of the trachea in lightly anaesthetized guinea pigs. Mechanical stimulation to the larynx or the bifurcation of trachea caused a stable cough response. The response was reproducible over 60 min, when stimulation was repeatedly applied at 20-min intervals. No significant difference was found between the amplitudes of the responses to mechanical stimulation of the larynx and of the tracheal bifurcation. Codeine, 10, 20 and 50 mg/kg, dose dependently depressed the coughs caused by larynx stimulation. The antitussive, however, failed to depress the cough caused by stimulation to the tracheal bifurcation, although a large dose, 50 mg/kg, significantly depressed the cough. In capsaicin-treated guinea pigs, codeine at 20 mg/kg significantly depressed the cough caused by stimulation to the tracheal bifurcation. The present results suggest that cough caused by mechanical stimulation to the larynx might be more sensitive to codeine treatment than cough caused by stimulation to the bifurcation of trachea. Furthermore, it is suggested that coughs caused by mechanical stimulation to both sites might consist of at least two components as regards their pharmacological nature.

Both beta 1- and beta 2-adrenoceptors are involved in mediating phosphatidylcholine secretion in rat type II pneumocyte cultures.

A primary culture of rat type II pneumocytes was used for the pharmacological and functional characterization of beta-adrenoceptor subtypes. The beta-adrenoceptor agonists, isoprenaline, dobutamine and procaterol concentration dependently increased the secretion of phosphatidylcholine. These effects were attenuated by propranolol. The effect of dobutamine was attenuated by atenolol, and that of procaterol by ICI 118,551. Isoprenaline-induced secretion was attenuated by the combination of the two blockers but not by each one alone. In conclusion, both beta 1- and beta 2-adrenoceptor subtypes mediate phosphatidylcholine secretion in rat type II pneumocytes.

Potentiation of phenobarbital-induced anticonvulsant activity by pipecolic acid.

Pipecolic acid (PA) is an intermediate of lysine metabolism in the mammalian brain. Recent findings suggest a functional connection of PA as neuromodulator in GABAergic transmission. Since many drugs are postulated to produce their effects by interaction with the central GABA system, the influence of PA on the anticonvulsant activity of phenobarbital was examined. Pretreatment of mice with 50 mg . kg-1 of PA potentiated the suppressing effects of the barbiturate on electrically and chemically induced convulsions. However, there was no potentiation of the behavioral effects and hypothermia induced by phenobarbital. PA itself had no or only little effect on the convulsions, motor function and rectal temperature when given in i.p. doses up to 500 mg . kg-1. Intraventricular administration of 500 microgram of PA also did not suppress either type of convulsion, although it produced ptosis, hypotonia, sedation and hypothermia. The results are discussed in relation to GABA system.

Effects of anesthetics on piperidine levels in mouse brain.

Piperidine is one of the biogenic amines possessing potent pharmacological activity. Recent interest has focused on its possible role as an endogenous hypnogenic substance. Using a mass fragmentographic technique with deuterium-labelled piperidine as an internal standard, piperidine concentrations in brains of waking and deeply anesthetized mice were analyzed to compare piperidine levels in the brain under distinctly different states of consciousness. A rapid and significant increase in piperidine concentrations was found in the brain but not in blood of mice anesthetized with any one of pentobarbital, urethane, ether and halothane. The results, showing that CNS depression is accompanied by accumulation of piperidine in the brain, are consistent with the idea that piperidine may have a close connection with the mechanisms controlling the level of consciousness.

Changes in brain piperidine levels under anesthesia: mass fragmentographic analysis.

Piperidine is a biogenic alicyclic amine possessing potent pharmacological activity. Interest has recently been focussed on its possible role as an endogenous hypnogenic substance. Using a mass fragmentographic technique with deuterium-labelled piperidine as an internal standard, the time relations of the change in brain levels of piperidine and the anesthetic activity of urethane were determined in mice. The brain piperidine level increased prior to the loss of the righting reflex and the elevated level declined prior to the reappearance of the reflex. The change in brain piperidine level correlated with neither that in spontaneous motility nor that in body temperature. The findings favor the idea that piperidine might at least partly regulate the level of consciousness.