gamma-Aminobutyric acid-induced modulation of acetylcholine release from the guinea pig lung

gamma-Aminobutyric acid (GABA) content was measured biochemically and the effect of GABA on the release of [3H]acetylcholine (ACh) was studied in strips of the guinea pig lung preloaded with [3H]choline. GABA contents were highest in the middle sections of the lung, as compared with proximal and distal areas. GABA evoked the release of [3H]ACh from the strips of the lung. The effect of GABA was mimicked by muscimol and antagonized by bicuculline and furosemide. Perfusion with Ca++-free medium and tetrodotoxin, but not nipecotic acid, inhibited the GABA- and muscimol-evoked release of [3H]ACh, thereby indicating that the released ACh was of neuronal origin. Diazepam and pentobarbital potentiated the muscimol-evoked [3H]ACh release. On the other hand, GABA reduced the KCl (40 mM)-evoked release of [3H]ACh in the presence of tetrodotoxin and bicuculline and baclofen mimicked the inhibitory effect of GABA. The effects of GABA and baclofen were not altered by alpha and beta adrenergic antagonists. These findings provide evidence for two types of GABA receptors in the lung of the guinea pig, and these receptors are involved in regulating the release of ACh.     

Effects of mitoxantrone on excitation-contraction coupling in guinea pig ventricular myocytes

The mechanisms of the inotropic effect of mitoxantrone (MTO), a synthetic dihydroxyanthracenedione derivative with antineoplastic activity, was investigated in guinea pig ventricular myocytes using whole-cell patch-clamp methods combined with fura-2 fluorescence and cell-edge tracking techniques. In right ventricular papillary muscles, 30 microM MTO increased isometric force of contraction as well as action potential duration (APD) in a time-dependent manner. The force of contraction was increased approximately 3-fold within 4 h. This positive inotropic effect was accompanied by a prolongation of time to peak force and relaxation time. In current-clamped single myocytes treated with 30 microM MTO for 30 min, an increase of cell shortening by 77% and a prolongation of APD by 19% was observed. Peak amplitude of the intracellular Ca(2+) transients was also increased by 10%. The contribution of APD prolongation to the enhancement of cell shortening induced by MTO was assessed by clamping control myocytes with action potentials of various duration. Prolongation of APD(90) (ADP measured at 90% of repolarization) by 24% led to an increase of cell shortening by 13%. When the cells were clamped by an action potential with constant APD, MTO still caused an increase of cell shortening by 59% within 30 min. No increase of the peak intracellular Ca(2+) transients, however, was observed under this condition. We conclude that both the APD prolongation and a direct interaction with the contractile proteins contributed to the positive inotropic effect of MTO.     

Effects of cyclosporine on chronic cytomegalovirus infection in the guinea pig

Following establishment of chronic guinea pig cytomegalovirus (GPCMV) infection, animals received by gavage either cyclosporine (CS), 20-33 mg/kg, or drug vehicle daily for 28 days. At sacrifice the frequency of CMV isolation from tissues of GPCMV-infected animals was 20% for the immunosuppressed group versus 12% for controls (difference, NS). GPCMV isolation rates from lymphoid tissues (thymus, spleen, cervical lymph nodes) of experimental animals was 7 of 69 (10%) vs. 0 of 42 (0%) from controls (p less than 0.05). Although CS immunosuppression was associated with increased rates of GPCMV isolation from lymphoid tissues of chronically infected animals, it was not accompanied by detectable systemic disease.     

Effects of intravitreal ropivacaine on retinal thickness and integrity in the guinea pig

Background:

Retrobulbar anesthesia is widely used for ocular surgery.Ocular complications are possible when retrobulbar anesthesia is accidentally injected intravitreally.

Objective:

The aim of this study was to determine the relative retinal toxicitiesof ropivacaine hydrochloride, a local anesthetic, using various concentrations in guinea pigs.

Methods:

This randomized, investigator-masked, experimental study wasconducted at the Department of Anesthesiology, Dicle University, Diyarbakir, Turkey. The right eyes of 18 guinea pigs were assigned to 1 of 3 treatment groups: 1%, 0.75%, or 0.5% ropivacaine. The right eye of each animal was injected intravitreally with 0.1 mL of 1%, 0.75%, or 0.5% ropivacaine. The left eye of each animal was injected with a balanced saline solution (control). The guinea pigs were euthanized 7 days after injection, and the retinal structures were examined using light microscopy. The total thickness of each retina was measured using an ocular micrometer.

Results:

No histologic abnormalities were observed in the control eyes.Retinal damage of most of the retinal section was seen in the eyes receiving study drug. The eyes injected with 0.5% ropivacaine had a generally intact appearance, with the exception of some atrophy and disorganization. Overall, the eyes injected with 1% ropivacaine had significantly more extensive retinal thinning compared with the eyes injected with 0.75% or 0.5% ropivacaine (both, P < 0.01). In the eyes injected with 0.75% or 1% ropivacaine, disorganization of the structure of the retinal layers and atrophy were noted on histopathology. The mean total thicknesses of the retina were significantly less in all ropivacaine-treated eyes compared with that in the controls (P < 0.001)

Conclusions:

In this small experimental study, ropivacaine had concentration-dependent toxic effects on guinea pig retinas.     

Release of acetylcholine mediated by cholecystokinin receptor from the guinea pig sphincter of Oddi

Mechanisms involved in the action of cholecystokinin octapeptide (CCK-8) on the isolated circular muscle of the guinea pig sphincter of Oddi were investigated. CCK-8 (10(-9) to 10(-6) M) caused a concentration-dependent contraction of the sphincter. Angiotensin II, bethanechol and serotonin also contracted this tissue. CCK-8 exerted the most potent effect. However, bradykinin, porcine motilin and norepinephrine failed to elicit any contraction. The CCK-8-induced contraction was inhibited by about 60% by atropine (3 X 10(-7) M) or tetrodotoxin (3 X 10(-7) M) but was not affected by hexamethonium (3 X 10(-5) to 10(-4) M) or phentolamine (3 X 10(-6) to 10(-5) M). Proglumide (3 X 10(-4) to 3 X 10(-3) M), a derivative of glutaramic acid, inhibited competitively the contraction by CCK-8. However, proglumide influenced neither the electrically elicited twitch contraction nor the bethanechol-induced contraction. CCK-8 evoked a concentration-related release of [3H]acetylcholine (ACh) from previously labeled stores. The CCK-8-evoked release of [3H]ACh was eliminated by tetrodotoxin and was inhibited, in a concentration-dependent manner, by proglumide. These results suggest that the contractile response to CCK-8 of the guinea pig sphincter of Oddi consists of a direct effect on the smooth muscle and an indirect effect mediated by ACh release from postganglionic parasympathetic neurons.     

Induced release of acetylcholine from guinea pig ileum longitudinal muscle-myenteric plexus by anatoxin-a.

Anatoxin-a (ANTX), a nicotinic agonist, has been shown to induce contraction of guinea pig ileum, which was abrogated by the muscarinic antagonist atropine and the nicotinic antagonists tubocurarine and hexamethonium. We showed here that the ganglionic nicotinic antagonist mecamylamine was a better inhibitor of the contraction of ileum induced by ANTX. The sodium channel blocker tetrodotoxin also abolished ANTX-induced contraction. In contrast, alpha-bungarotoxin, the muscle type nicotinic receptor blocker, had no effect on ANTX-induced contraction of guinea pig ileum. Longitudinal muscle-myenteric plexus prepared from guinea pig ileum, labeled with [3H]choline and then incubated with ANTX was shown for the first time to release [3H]acetylcholine (ACh) in a dose-dependent manner. Pretreatment of longitudinal muscle-myenteric plexus with tubocurarine, hexamethonium or mecamylamine blocked ANTX-induced release of [3H]ACh. In contrast, atropine was without effect. Mecamylamine was the most potent antagonist. As observed in ileum contraction, tetrodotoxin completely and potently blocked the release of [3H]ACh induced by ANTX. Neither alpha-bungarotoxin nor the neuromuscular junction blockers conotoxin G1 or M1 could inhibit the [3H]ACh release. Taken together, these results suggested that ANTX activated nicotinic receptors on ganglionic interneurons to trigger a release of ACh, which next stimulated muscarinic receptors and induced ileum contraction.     

Pharmacological properties of nicotinic acetylcholine receptors expressed by guinea pig small intestinal myenteric neurons

The electrophysiological and pharmacological properties of nicotinic acetylcholine receptors (nAChRs) were studied in guinea pig small intestinal myenteric neurons maintained in culture or in acutely isolated preparations. Acetylcholine and nicotine caused inward currents that desensitized in approximately 4 s. The current-voltage (I-V) relationship rectified inwardly with a reversal potential near 0 mV. The agonist rank order potency was 1,1-dimethyl-4-phenyl-piperazinium > acetylcholine = nicotine > cytisine. Agonist-induced currents were blocked by nAChR antagonists with a rank order potency of mecamylamine > hexamethonium > dihydro-beta-erythroidine (DHbetaE); mecamylamine and DHbetaE exhibit high potency at beta4 and beta2 subunit-containing nAChRs, respectively. alpha-Bungarotoxin (0.1 microM) or alpha-methyllycaconitine (0.1 microM), antagonists that block nAChRs containing alpha7 subunits, did not affect acetylcholine-induced responses. Immunohistochemical studies revealed that nearly every neuron in culture was labeled by an antibody (mAb35) that recognizes nAChR alpha3 and alpha5 subunits. Antibodies selective for alpha3, alpha5, or beta2 subunits also stained most neurons, whereas an alpha7 subunit antibody revealed very few neurons. In neurons in the intact myenteric plexus from newborn and adult guinea pigs, local application of acetylcholine (1 mM) and cytisine (1 mM) caused similar amplitude depolarizations, and these responses were blocked by nAChR antagonists with a rank order potency of mecamylamine > hexamethonium > DHbetaE. These data indicate that myenteric neurons maintained in culture predominantly express nAChRs composed of alpha3, alpha5, beta2, and beta4 subunits. These subunits may be in a homogeneous population of receptors with unique pharmacological properties, or multiple receptors of different subunit composition may be expressed by individual neurons.     

Effects of forssman antibody on renal function of the guinea pig

When guinea pigs were injected with rabbit anti-sheep red blood cell antiserum (Forssman antibody) by the intra arterial (i.a.) route (abdominal aorta at the level of the renal arteries), they developed renal disfunction. A diminished glomerular filtration rate associated with a 27% increase in the fractional excretion of K+ was observed. No alterations were noted in the fractional excretion of water and NA+.     

盐酸雷诺秦对豚鼠乳头肌动作电位及收缩力的影响

目的通过盐酸雷诺秦对离体豚鼠心室乳头肌动作电位及收缩力的影响,探讨其抗心律失常及心肌缺血的机制。方法健康成年豚鼠18只,随机分为H2O2(200 mmol.L-1)组、雷诺嗪(10 mmol.L-1)+H2O2组和TTX(2 mmol.L-1)+H2O2组,每组6只,采用给药前后自身对照的方法观察雷诺秦对豚鼠乳头肌的作用。结果H2O2可使对照组动作电位APD50、APD90明显增加(P0.001),心肌收缩力降低(P0.05),雷诺嗪可抑制H2O2引起的动作电位时程APD50、APD90的延长(P0.05和P0.01),但作用弱于TTX(P0.05和P0.001),雷诺嗪、TTX可改善H2O2引起的心肌收缩力降低。结论盐酸雷诺嗪可降低H2O2引起的豚鼠乳头肌动作电位动时程的增加和增强心肌收缩力,作用和TTX结果相似。     

豚鼠离体右心房自律性及收缩特性与葛根素的影响

目的:观察葛根素对豚鼠离体右心房自律性及收缩特性的影响,并探讨其作用机制。方法:实验于2005-07在赣南医学院药理实验室(省级重点实验室)完成。健康豚鼠6只,用锤子击打头部至昏迷,剖开胸腔迅速取出心脏,在持续充纯氧的条件下,分离右心房。将右心房肌悬挂在含20mLKrebs液的麦氏浴槽中,恒温(37±0.5)℃,持续通入纯氧。右心房稳定45min后,测定其正常自动节律、收缩幅度、收缩速度和舒张速度作为给药前对照,然后累积加入葛根素(终浓度为0.0036,0.012,0.036,0.12,0.36,1.2,3.6mmol/L),每次给药间隔5min,测定其自动节律、收缩幅度、收缩速度、舒张速度的变化。结果:葛根素可降低豚鼠离体右心房的自律性、收缩幅度、收缩速度、舒张速度,且有明显的剂量依赖性。结论:葛根素可抑制豚鼠离体右心房的自律性,明显降低豚鼠离体右心房的收缩幅度、收缩速度、舒张速度,且有明显的剂量依赖性,其作用机制可能与抑制钙离子通道及钠离子通道有关。     

Nicotinic acetylcholine receptors at sites of neurotransmitter release to the guinea pig intestinal circular muscle

Experiments were designed to test the hypothesis that nicotinic acetylcholine receptors (nAChRs) are present at sites of neurotransmission to the guinea pig ileum circular smooth muscle. Circular smooth muscle preparations, from which the myenteric plexus had been removed (circular muscle-axon preparation), were used for this purpose. Nicotine and dimethylphenyl piperazinium iodide (10-100 microM) induced contraction of the circular smooth muscle. Agonist-induced contraction was inhibited by 1 microM scopolamine and abolished in the combined presence of 1 microM scopolamine and 0. 3 microM CP 96,345-01, a neurokinin-1 receptor antagonist. Contractions induced by electric field stimulation (30 pulses, 0.5 ms, 70 V, 10 Hz) were abolished by 0.3 microM tetrodotoxin (TTX); in contrast, agonist-induced contraction was attenuated but not abolished by 0.3 microM TTX. Mecamylamine (3 or 30 microM), an nAChR antagonist, blocked agonist-induced contractions. Frequency-response curves for both "ON and "OFF electric field stimulation contractions were abolished by the combined presence of 1 microM scopolamine and 0.3 microM CP 96,345-01 or by 0.3 microM TTX. At stimulation frequencies greater than 2 Hz, the ON contraction was increased in the presence of 100 microM nitro-L-arginine. Mecamylamine (3 microM) was used to block the stimulatory prejunctional nAChRs located near sites of neurotransmitter release to the circular smooth muscle; however, ON and OFF contractions were not affected by mecamylamine. Although the prejunctional nAChRs are not targets for endogenously released acetylcholine under the conditions tested here, these receptors may be targets for the development of new prokinetic agents.     

Effects of repeated 3,4-methylenedioxymethamphetamine administration on neurotransmitter efflux and sensory-evoked discharge in the ventral posterior medial thalamus

3,4-Methylenedioxymethamphetamine (MDMA) is known to enhance tactile sensory perception, an effect that contributes to its popularity as a recreational drug. The neurophysiological basis for the effects of MDMA on somatosensation are unknown. However, MDMA interactions with the serotonin transporter (SERT) and subsequent enhancement of serotonin neurotransmission are well known. The rat trigeminal somatosensory system receives serotonergic afferents from the dorsal raphe nucleus. Because these fibers express SERT, they should be vulnerable to MDMA-induced effects. We found that administration of a challenge injection of MDMA (3 mg/kg i.p.) after repeated MDMA treatment (3 mg/kg per day for 4 days) elicits both serotonin and norepinephrine efflux in the ventral posterior medial (VPM) thalamus of Long-Evans hooded rats, the main relay along the lemniscal portion of the rodent trigeminal somatosensory pathway. We evaluated the potential for repeated MDMA administration to modulate whisker-evoked discharge of individual neurons in this region. After surgically implanting stainless steel eight-wire multichannel electrode bundles, we recorded spike train activity of single cells while activating the whisker pathway using a piezoelectric mechanical stimulator. We found that repeated MDMA administration increased the spontaneous firing rate but reduced both the magnitude and duration of whisker-evoked discharge in individual VPM thalamic neurons. The time course of drug action on neuronal firing patterns was generally consistent with fluctuations in neurotransmitter efflux as shown from our microdialysis studies. On the basis of these results, we propose that single use and repeated administration of MDMA may "distort," rather than enhance, tactile experiences in humans, in part, by disrupting normal spike firing patterns through somatosensory thalamic relay circuits.     

Stimulation of acetylcholine release from myenteric neurons of guinea pig small intestine by forskolin and cyclic AMP

Forskolin, an activator of adenylate cyclase, was used to examine the regulation of [3H]acetylcholine (ACh) release by cyclic AMP (cAMP)-related mechanisms in myenteric plexus-longitudinal muscle preparations of guinea pig small intestine. Forskolin evoked a dose-related increase in [3H]ACh release. Both dibutyryl-cAMP and 8-Br-cAMP significantly elevated [3H]ACh secretion. In the presence of phosphodiesterase inhibitors (theophylline and 3-isobutyl-1-methylxanthine), the basal [3H]ACh output was increased. There was a significantly greater stimulation when forskolin was used to incite endogenous cAMP synthesis and phosphodiesterase inhibitors were simultaneously applied to prevent cAMP breakdown. The enhancement of forskolin-stimulated release by theophylline or 3-isobutyl-1-methylxanthine strongly implicates a synergistic interaction between the two. These findings suggest that forskolin acts to increase ACh release by a modulation of endogenous cAMP and further support a cAMP-mediated mechanism in the secretion of ACh from myenteric cholinergic neurons.     

Interaction of cholecystokinin and diazepam: effects on brain monoamines

An antagonism between cholecystokinin (CCK) peptides and benzodiazepines (BZD) has been described in various paradigms. We sought to determine whether CCK and BZD are also antagonistic in their effects on brain neurotransmitter levels in the rat. No effect on the noradrenergic system was induced in any brain area by CCK 8 S and diazepam alone or in combination. Administered alone, sulfated CCK octapeptide (CCK 8 S) (5 micrograms/kg ip) and diazepam (5 mg/kg ip) were found to decrease DOPAC levels in the cortex and to induce 5-hydroxy-tryptamine accumulation in the hippocampus. When administered together, these variations were no longer observed. However, a slight tendency by each substance to decrease 3-methoxy-tyramine levels in the striatum, became significant when given in association. The differences in CCK-BZD interactions observed in the striatum, cortex and hippocampus suggest that different mechanisms of action are involved. The addition of the effects occurring in the striatum might involve a GABA-ergic mechanism.