Teruko Takeo

Axon-sparing lesion of the preoptic area enhances receptivity and diminishes proceptivity among components of female rat sexual behavior

Stereotaxic infusion of ibotenic acid deleted neurons in the medial preoptic area (POA) in the ovariectomized female rats. A well-circumscribed lesion was infiltrated by astrocytes; local axons of passage were spared. Following estrogen priming and progesterone supplement, the females with the lesion had higher lordosis quotients than the vehicle-infused controls, when males successfully mounted them. On the other hand, the treatment did not induce solicitation in females with the lesion nor reduced their rejection of male partners. Meanwhile, gradual and persistent suppression of the lordosis reflex followed electrical stimulation through electrodes placed in the POA lesion. Except that the females with the POA lesion needed less estrogen to obtain comparable prestimulation quotients with the controls, the lesioned and control animals responded similarly to the stimulation. Because an adjunct neural transection dorsal to the POA lesion abolished the stimulus-bound suppression of lordosis, the effect was due to the activation of axons of passage that presumably descend from the septum. It is concluded that the POA is the major target for estrogen in eliciting proceptive behavior; local POA neurons as well as septal efferents appear to inhibit the lordosis reflex, the principal receptive component in female rat sexual behavior.

Suppression of the lordosis reflex of female rats by efferents of the medial preoptic area

In freely moving, estrogen- and progesterone-treated ovariectomized female rats, electrical stimulation of the medial preoptic area, which had been isolated dorsally from the septum by a horizontal knife cut (roof cut), caused immediate interruption of the lordosis reflex, a principal component of female reproductive behavior in this species. Neither the knife cut nor the stimulation affected their proceptive interactions with the males. Lordosis was interrupted in a graded manner in response to increased stimulus intensity, with a threshold at 30 microA. The optimal frequency was at 50-100 Hz. Lordosis was reinstated promptly by the termination of current application. The rapid time course distinguished the stimulation effect in the rats with the roof cut from that in the intact control animals, which was slow in its onset and recovery. In the sham-operated animals, an adjunct bilateral cut of the stria terminalis was sufficient to restore the rapid response as in the roof-cut animals. The roof-cut animals were sensitive to lower doses of estrogen than those without the knife cut; therefore, the preoptic area appears to be a separate entity from the septum in the inhibitory control of lordosis. The elimination of facilitatory neural components for this reflex, which enter the preoptic area dorsally via the stria terminalis, might be responsible for the prompt and exaggerated stimulation effect in the roof-cut animals.

GLP-I(7–36) Amide Augments Ba2+ Current Through L-Type Ca2+ Channel of Rat Pancreatic β-Cell in a cAMP-Dependent Manner

The whole-cell patch-clamp method was used to examine the effect of glucagon-like peptide I (GLP-I)(7–36) amide on the activation process of L-type Ca2+ channels of rat pancreatic β-cells. After depolarization, GLP-I (1–100 nmol/l) caused action potentials in cells exposed to a glucose-free solution for 10 min. The percentage of cells producing action potential depended on the concentration of GLP-I. In some cells, GLP-I caused action potentials without the prior depolarization of the membrane. In cells exposed to the glucose-free solution for longer than 30 min, or in cells that were deprived of ATP by a means of the conventional whole-cell configuration, GLP-I (20 nmol/l) did not cause the electrical excitation. Application of GLP-I augmented the maximum Ba2+ current (IBa) through L-type Ca2+ channels and shifted the current voltage curve to the left. Values of changes in the maximum IBa depended on GLP-I concentration. Application of dibutyryl cAMP (dbcAMP, 1 mmol/1) also augmented IBa. In cells pretreated with Rp-cAMP, dbcAMP did not change the magnitude of IBa. Also in cells pretreated with Rp-cAMP, GLP-I failed to augment IBa. These results suggest that in pancreatic β-cells, GLP-I, by a cAMP-dependent mechanism, increases opening of L-type Ca2+ channels. cAMP-dependent augmentation of Ca2+ entry as well as cAMP production itself by GLP-I plays a crucial role in controlling insulin secretion.

Diametrically opposite effects of estrogen on the excitability of female rat medial and lateral preoptic neurons with axons to the midbrain locomotor region.

Electrical stimulation of the midbrain locomotor region (MLR) in 76 ovariectomized, urethan-anesthetized female rats elicited antidromic action potentials in 252 preoptic neurons. Thresholds and refractory periods for the activation ranged from 60 to 1550 microA and 1.3 to 5.0 ms, respectively. The probability distribution for the peak-to-peak amplitude (2-14 mV) or the overall duration (0.7-4.4 ms) was bell-shaped, whereas that for the latency (1.8-33.5 ms) was distinctively bimodal with a division at 12.0 ms. Two groups of preoptic neurons of a similar soma size therefore project to the MLR presumably via different routes. In 121 neurons with latencies < or = 12.0 ms, estrogen lowered the antidromic activation thresholds (nested analysis of variance, P < 0.02), but 131 neurons with latencies > 12.0 ms had their thresholds increased (P < 0.005) and refractory periods prolonged (P < 0.02) by estrogen. Even though both overlapped in part, many potentials with the shorter latencies were recorded from the medial part of the lateral preoptic area (mLPO), lateral to the recording sites of the longer-latency potentials in the medial preoptic area (MPO). The observed antagonistic effects of estrogen on the two groups of preoptic neurons with axons to the MLR may contribute to increased locomotor activity in female rats in estrus.

Iptakalim modulates ATP-sensitive K+ channels in dopamine neurons from rat substantia nigra pars compacta

Iptakalim, a novel cardiovascular ATP-sensitive K+ (KATP) channel opener, exerts neuroprotective effects on dopaminergic (DA) neurons against metabolic stress-induced neurotoxicity, but the mechanisms are largely unknown. Here, we examined the effects of iptakalim on functional KATP channels in the plasma membrane (pm) and mitochondrial membrane using patch-clamp and fluorescence-imaging techniques. In identified DA neurons acutely dissociated from rat substantia nigra pars compacta (SNc), both the mitochondrial metabolic inhibitor rotenone and the sulfonylurea receptor subtype (SUR) 1-selective KATP channel opener (KCO) diazoxide induced neuronal hyperpolarization and abolished action potential firing, but the SUR2B-selective KCO cromakalim exerted little effect, suggesting that functional KATP channels in rat SNc DA neurons are mainly composed of SUR1. Immunocytochemical staining showed a SUR1-rather than a SUR2B-positive reaction in most dissociated DA neurons. At concentrations between 3 and 300 μM, iptakalim failed to hyperpolarize DA neurons; however, 300 μM iptakalim increased neuronal firing. In addition, iptakalim restored DA neuronal firing during rotenone-induced hyperpolarization and suppressed rotenone-induced outward current, suggesting that high concentrations of iptakalim close neuronal KATP channels. Furthermore, in human embryonic kidney 293 cells, iptakalim (300-500 μM) closed diazoxide-induced Kir6.2/SUR1 KATP channels, which were heterologously expressed. In rhodamine-123-preloaded DA neurons, iptakalim neither depolarized mitochondrial membrane nor prevented rotenone-induced mitochondrial depolarization. These data indicate that iptakalim is not a KATP channel opener in rat SNc DA neurons; instead, iptakalim is a pm-KATP channel closer at high concentrations. These effects of iptakalim stimulate further pharmacological investigation and the development of possible therapeutic applications.

Intracellular Ca2+ Modulation of ATP-Sensitive K+ Channel Activity in Acetylcholine-Induced Activation of Rat Pancreatic β-Cells

We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.

Opposite Effects of Two Resveratrol (trans-3,5,4′-Trihydroxystilbene) Tetramers, Vitisin A and Hopeaphenol, on Apoptosis of Myocytes Isolated from Adult Rat Heart

It has been reported that resveratrol (trans-3,5,4′-trihydroxystilbene) from Vitis plants has various cardioprotective effects. Vitis plants also include various resveratrol tetramers. The aim of our study is to clarify the pharmacological properties of resveratrol tetramers. We isolated two resveratrol tetramers as major products of Vitis plants. One is vitisin A, a complex of two resveratrol dimers, (+)-ϵ-viniferin and ampelopsin B, and the other is hopeaphenol, composed of 2 mol ampelopsin B. Vitisin A (30–300 nM) unexpectedly dose-dependently facilitated swelling and depolarization of mitochondria and cytochrome c release from mitochondria, which are indices of cardiomyocyte apoptosis. Furthermore, vitisin A induced apoptosis in the primary culture of adult rat ventricular myocytes. On the other hand, hopeaphenol (1–10 μM) dose-dependently inhibited Ca2+ (30 μM)-induced mitochondrial depolarization and cytochrome c release from mitochondria but had not affected mitochondrial swelling. Moreover, hopeaphenol inhibited vitisin A-induced apoptosis. In structural and functional studies, we further confirmed that vitisin B, one of the resveratrol tetramers having (+)-ϵ-viniferin unit, induces mitochondrial swelling and cytochrome c release from mitochondria like vitisin A and that vitisifuran A, one of the resveratrol tetramers having the ampelopsin B unit, inhibits Ca2+-induced cytochrome c release from mitochondria like hopeaphenol. These results show that resveratrol tetramers have at least two opposite effects on cardiomyocytes; the one having the (+)-ϵ-viniferin unit induces cardiomyocyte apoptosis, and the other having ampelopsin B but not (+)-ϵ-viniferin unit inhibits it.

Interruption of the lordosis reflex of female rats by ventral midbrain stimulation

The lordosis reflex, dorsiflexion of the vertebral column, is an estrogen-dependent, essential element of female sexual behavior in rodents. Unilateral electrical stimulation of the midbrain ventral tegmental area through a chronically implanted electrode in freely moving, estrogen-primed ovariectomized female rats caused a rapid and strong suppression of the lordosis reflex in response to either male mounts or manual cutaneous stimuli. The interruption occurred in a graded manner to increased stimulus intensity, with a threshold at 30 microA. The optimal frequency was at 75-125 Hz. After the termination of electrical stimulation, lordosis performance returned promptly to the pre-stimulation level. No aversive response accompanied the blockade of lordosis. Electrical stimulation specifically blocked lordosis, without disrupting the proceptive components of female sexual behavior. In 10 animals tested, concomitant injection of dopamine receptor blocker pimozide tended to offset the effects of electrical stimulation in 2 cases. Interruption of the lordosis reflex might be mediated by projections from the ventral tegmental area, which activate a descending pathway inhibitory to the lordosis reflex arc at or below the lower brain stem.

Thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse

Modulation of the agonist‐specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch‐clamp perforated whole‐cell recording to measure the calcium‐dependent chloride current (I C1(Ca2+)). 1 μM thimerosal, which fails to evoke Ca2+ oscillation f alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist‐specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3‐induced Ca2+ release.

Protein kinase C-dependent and -independent inhibition of Ca2+ influx by phorbol ester in rat pancreatic β-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic β-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca2+ concentration ([Ca2+]i) elevated by the glucose stimulation when tested in isolated rat β-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized β-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca2+ currents through L-type Ca2+ channels. BIM prevented the PMA inhibition of the Ca2+ currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic β-cells, defeating its own inhibition of the Ca2+ influx through L-type Ca2+ channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.