GABA evoked ACh release from isolated guinea pig ileum

To identify the target cells of GABAergic neurons located in the myenteric plexus, the action of γ-aminobutyric acid (GABA) on the release of acetylcholine (ACh) and on the contractions was studied using the isolated guinea pig ileum. GABA evoked a release of ³H-ACh from the contracting ileum, under conditions of loading with ³H-choline. As both the GABA-evoked release of ³H-ACh and the contractions were inhibited by bicuculline, tetrodotoxin and furosemide, but not by hexamethonium, this release seems to be evoked through GABA receptors which are bicuculline sensitive and associated with the Cl- ion channel.     

Mathematical description of regenerative potentials recorded from circular smooth muscle of guinea pig antrum

Regenerative potentials evoked by intracellular current injection in single bundles of circular smooth muscle taken from guinea pig antrum have the characteristics of the secondary regenerative component of the slow wave occurring in the same muscle layer. Such regenerative depolarizations might result from a mechanism that responds to membrane polarization with a delayed increase in the rate of production of unitary potentials detected in this tissue. To test this possibility, a two-stage reaction leading to the formation of an intracellular messenger was proposed. The first forward reaction was voltage-dependent, in the manner described by the Hodgkin-Huxley transient Na conductance formalism, allowing simulation of anode break excitation, stimulus threshold strength-duration characteristics, and refractory behavior. A conventional dose-effect relationship was proposed to describe the dependence of the mean rate of discharge of unitary potentials on messenger concentration. Unitary potentials were modeled as unitary membrane conductance modulations with an empirically derived amplitude distribution and Poisson-distributed intervals. The model reproduces a range of spontaneous and evoked membrane potential changes characteristic of antral circular muscle bundles.     

Early effect of glucose and oxygen deprivation on the spontaneous acetylcholine release from the myenteric plexus of the guinea pig ileum

The early effects of glucose and oxygen deprivation on the spontaneous acetylcholine output from the myenteric plexus - longitudinal muscle preparation of the guinea pig ileum were studied using an incubation chamber that permitted rapid sample collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine collection in 2-min intervals. Glucose deprivation or hypoxia resulted in a gradual decline in rate of spontaneous acetylcholine output. However, glucose deprivation plus hypoxia caused an acceleration in acetylcholine output within 10-15 min, which attained a rate seven times greater than observed under normal conditions. Recovery of low resting rates was obtained by reintroduction of oxygen and glucose into the bath medium. Neither morphine (2.7 x 10(-5) M) nor tetrodotoxin (1.6 x 10(-6) M) prevented the increase in acetylcholine output induced by energy deprivation. The substitution of Ca2+ by Mg2+, in the presence of EGTA, greatly reduced the acetylcholine output induced by energy deprivation. However, a small transitory output of acetylcholine was observed under these conditions which was resistant to tetrodotoxin and ot affected by depolarizing amounts of K+. The transitory output was repeatable by reintroduction of glucose and oxygen to the Ca2+-free medium with subsequent return to conditions of hypoxia and glucose deprivation. These results suggest that energy deprivation initially stimulates normal acetylcholine secretion by (a) increasing Ca2+ influx across the plasma membrane and (b) mobilizing an intracellular Ca2+ poll. This implies that processes involved in maintenance of normal low transmitter release are more sensitive to energy lack than the neurosecretion process itself.     

Calcium dependence of prostaglandin release from the guinea pig Taenia coli

We studied the calcium dependency of the stimulation of prostaglandin synthesis which occurs when perfusing strips of guinea pig Taenia coli with potassium-free media. Stimulation was rapidly reversed by removal of extracellular Ca from the bathing solution. The Ca ionophore A23187 markedly stimulated prostaglandin E2 synthesis, an effect that is dependent on the presence of extracellular Ca. Prostaglandin E2 production in strips in potassium-deficient media was also sensitive to increases in extracellular Ca, and was augmented at concentrations of 7-15 mM. In strips which had been incubated with [3H]arachidonic acid, exposure to potassium-free media caused an increased release of [3H]arachidonic acid and [3H]prostaglandin E2. Release of these labeled compounds with the strips in potassium-free media was further augmented by increasing extracellular [Ca2+] from 2.5 to 10 mM. Treatment with the Ca antagonist agent verapamil did not influence activation of prostaglandin synthesis by potassium-deficient media. The presence of Mn2+ of Ba2+ had similar effects on prostaglandin synthesis, although they had opposite effects on mechanical activity. We conclude that a plasma membrane associated Ca pool is involved in activation of phospholipid metabolism which results in release of esterified arachidonic acid and subsequent prostaglandin synthesis. This Ca pool is in rapid equilibrium with extracellular Ca, is not influenced by cytoplasmic Ca, and is not related to Ca involved in Ca gating in the surface membrane. These data also indicate dissociation between processes involved in muscle contraction and activation of prostaglandin synthesis.     

Desensitization of isolated smooth muscle cells from guinea pig taenia caecum to acetylcholine

The role of tissue organization of smooth muscle in short-term desensitization to acetylcholine (ACh) was examined by studying the desensitization of isolated single cells from guinea pig taenia caecum. Cells were isolated by collagenase digestion. The conditions during cell isolation were adjusted to obtain cells that showed repeated contractions. The cells contracted on treatment with 10(-7)-10(-6) M ACh, showing an all-or-none response. Desensitized cells also showed an all-or-none response but required a higher concentration of ACh for induction of contraction; i.e., the magnitude of their maximal response was not changed appreciably but the threshold concentration of ACh for their contraction was raised. Incubation of the whole tissue with 10(-4) M ACh for 10 min also caused desensitization. This desensitization was accompanied by reduction of the contractile response at intermediate concentrations. The mode of desensitization of isolated cells determined from the average response of the isolated cells was almost the same as that of whole muscle. It is concluded that the desensitization occurred in each cell irrespective of its tissue organization and that the desensitization was due to an increase of the threshold for contraction to ACh of each cell.     

Action of thiamine on auditory evoked potentials in the guinea pig

A technique is proposed for quantifying the effects of physiologically active substances at the periphery of the auditory analyzer. It was found that applying 1×10^–11 to 1×10^–3 M thiamine to the membrane of guinea pig cochlear round window (fenestra rotunda) produces a rise in the amplitude and a reduction in the latency of the N₁ and N₂ components of auditory nerve action potentials, waves I and II of brainstem auditory evoked potentials occurring in response to an acoustic stimulus. It is suggested that this effect is produced by facilitated synaptic transmission at synapses between hair cells and spiral ganglia neurons under the action of thiamine penetrating into the cochlea.A. V. Palladin Institute of Biochemistry, Academy of Sciences of the Ukrainian SSR, Kiev. A. I. Kolomiichenko Research Institute of Otolaryngology, Ministry of Public Health of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 654–660, September–October, 1986.     

Neuropeptide release from isolated myenteric nerve endings derived from the guinea pig myenteric plexus.

Isolated myenteric nerve varicosities prepared from the myenteric plexus of the guinea pig ileum were investigated as a suitable model system with which to study the release of several neuropeptide-like immunoreactivities (-LI). Basal release of substance P-LI, neurokinin A-LI, Leu-enkephalin-LI and Met-enkephalin-LI was determined, and clear depolarization-induced release of the enkephalin-LI's and neurokinin A-LI was obtained using this preparation, providing further support for their roles as putative mediators in the enteric nervous system. Evoked-release of these peptides was dependent on the presence in the incubation mixture of certain antagonists to known endogenous neuronal mediators. In the absence of such antagonists, no unequivocal evidence of release was seen. Clear evoked release of Leu-enkephalin-LI occurred only in the presence of the adenosine receptor antagonist 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX), atropine and naloxone. Release of Met-enkephalin-LI occurred in the presence of either atropine or naloxone. The release of neurokinin A-LI was evident in the presence of DPSPX. These findings suggest the existence of either distinct subpopulations of nerve varicosities or distinct neuronal pools containing each peptide and that these peptides may be under differential regulation by endogenous inhibitory mediators. It is concluded that, under suitable conditions, isolated myenteric nerve varicosities provide a useful model system for the study of release, and the modulation of release, of endogenous neuropeptides.     

Simultaneous measurement of glutamate and dopamine release from isolated guinea pig cochlea

Glutamate is proved to be a neurotransmitter in the mammalian cochlea, transmitting signals between the inner hair cells and the afferent cochlear nerve terminals. The transmission in this synapse is modulated by the lateral olivocochlear efferent fibers by releasing dopamine and other neurotransmitters. This study undertakes to measure simultaneously the release of dopamine and glutamate from isolated guinea pig cochleae. We combined the in vitro microvolume superfusion method, that uses liquid scintillation analysis, to measure [3H]dopamine with high pressure liquid chromatography (HPLC) to determine the glutamate content of the superfusate at rest and during stimulation. The release of both neurotransmitters was significantly increased when electrical field stimulation was applied at a 10 Hz rate. The nonselective sodium-channel inhibitor tetrodotoxin (TTX) at 1 microM completely blocked the effect of stimulation, indicating the neural origin of both dopamine and glutamate. The dopamine receptor antagonist sulpiride at 100 microM and the dopamine receptor agonist bromocriptine at 20 microM did not change the release of glutamate. In contrast, both bromocriptine and sulpiride significantly increased the stimulation-evoked release of dopamine. The effect of sulpiride is most likely due to the blockade of dopamine autoreceptor. Possible explanations why bromocriptine increased the release include: (1) its partional agonist activity; (2) desensitizations of dopamine autoreceptors; or (3) the higher D1 receptor activity of bromocriptine than sulpiride. This study could provide further insights about the role of dopamine and glutamate in cochlear neurotransmission.     

The distribution of acetylcholine and choline in guinea pig heart

The regional distributions of acetylcholine (ACh) and choline (Ch) in the guinea pig heart were investigated with a pyrolysis-mass fragmentography technique. Using ACh as a marker for cholinergic neurons, we have described a pattern of parasympathetic innervation in the guinea pig heart. This distribution is very similar to that suggested by studies using several different cholinergic indicators in various species. Atrial areas receive richer parasympathetic innervation than ventricular areas, with the right portions receiving more than the left. The nodal areas were the most abundantly innervated regions examined. Ch content is not a good indicator for cholinergic innervation as the regional distribution of ACh and Ch throughout the guinea pig heart are not strongly associated.     

The auditory evoked potentials of the brain stem in the guinea pig

The examination of the standard waves' amplitude and latency of the brain stem auditory evoked response (BAEP) was performed in 20 guinea pigs (males and females, weighing 250 to 300 g). According with the relative loudness of stimuli (90, 70, 50, 30, 10 dB SPL), the latency of BAEP waves was larger (t1 = 0.2 msec), but the conductance time between P1 to P5 was constant (3.1 to 3.6 msec). The highest wave of BAEP was P2 with an amplitude: 90 dB SPL, U = 6.5 +/- 1.2 microV; 70 dB SPL, U = 4.3 +/- 1.0 microV; 50 dB SPL, U = 3.5 +/- 0.6 microV; 30 dB SPL, U = 2.0 +/- 0.4 microV.     

The influence of L-NG-nitro-arginine on field stimulation induced contractions and acetylcholine release in guinea pig isolated tracheal smooth muscle

The interaction between parasympathetic and inhibitory non-adrenergic, non-cholinergic nerves in tracheal smooth muscle was investigated by determining the effects of the NO-synthase inhibitor L-NG-nitro-arginine (L-NOARG) on contractions and the associated acetylcholine release elicited by field stimulation of the muscle. At frequencies above 2Hz contractile responses to field stimulation were potentiated by L-NOARG (50 microM). alpha-chymotrypsin pre-treatment potentiated contractile responses at all frequencies, but the effects of L-NOARG were unaltered. The effect of L-NOARG on responses to 5Hz electrical stimulation was not mimicked by D-NOARG, was reversed by L-, but not D-arginine and was unaffected by epithelium removal. L-NOARG did not affect responses to exogenous acetylcholine nor the overflow of 3H from tissues previously loaded with [3H]-choline. It is therefore concluded that field stimulation of tracheal smooth muscle induces the release of an endogenous nitrate, which, by an inhibitory action on smooth muscle, functionally antagonises the concomitantly released parasympathetic neurotransmitter.     

Prostaglandin release by slow reacting substance from guinea pig and human lung tissue

Slow reacting substance (SRS) injected into the pulmonary artery released prostaglandin E (PGE) and F_2α (PGF_2α) and the 15-keto-13, 14-dihydro PG metabolites from non-sensitized and ovalbumin sensitized, isolated, perfused guinea pig lungs. PGs were also released from lungs incubated with SRS. Sensitized lungs released more PGs in both types of preparations. Indomethacin inhibited the effect of SRS. Passively sensitized human lung fragments, in parallel to guinea pig lung, released PGE, PGF_2α and the metabolites when incubatted with SRS or antigen. In experiments, SRS and arachidonic acid given intravenously increased the airway insufflation pressure in anesthetized guinea pigs. These effects, but not the action of injected PGF_2α and histamine, were abolished by indomethacin. The results indicate that one of the modes of SRS action is by release of PGs, and are consistent with the hypothesis that PGs are predominantly “secondary” mediators (in the temporal sense) of the antigen-antibody reaction.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Calcium-induced calcium release mechanism in guinea pig taenia caeci

Fura-2 was used to measure the amount of Ca released from the intracellular Ca store of a saponin-skinned smooth muscle fiber bundle of the guinea pig taenia caeci (width, 150-250 microns) placed in a capillary cuvette at 20-22 degrees C. The amount of Ca actively loaded into the store was assayed when released by the application of 50 mM caffeine and/or 10 microM inositol 1,4,5-trisphosphate (IP3) in the absence of ATP, and was found to have a biphasic dependence on the loading [Ca2+] with a peak near pCa 6. After Ca loading at pCa 6, IP3 released almost all the releasable Ca, whereas caffeine discharged Ca from only approximately 40% of the store. The maximum amount of Ca in the store was some 220 mumol/liter cell water. Ca in the caffeine-releasable store was released approximately exponentially to zero with time when Ca2+ was applied in the absence of ATP, and the rate constant of the Ca-induced Ca release (CICR) increased steeply with the concentration of Ca2+ applied. Increase in [Mg2+] (0.5-5.0 mM) or decrease in pH (7.3-6.7) shifted the relation between pCa and the rate of CICR roughly in parallel toward the lower pCa. An adenine nucleotide increased the rate of the CICR, but it did not change the range of effective [Ca2+]. 5 mM caffeine greatly enhanced the CICR mechanism, making it approximately 30 times more sensitive to [Ca2+]. However the drug had no Ca-releasing action in the absence of Ca2+. Procaine in millimolar concentrations inhibited the rate of the CICR. These properties are similar to those of the skeletal muscle CICR and ryanodine receptor channels. Rates of the CICR under a physiological ionic milieu were estimated from the results, and a [Ca2+] greater than 1 microM was expected to be necessary for the activation of the Ca release. This Ca sensitivity seems too low for the CICR mechanism to play a primary physiological role in Ca mobilization, unless assisted by other mechanisms.     

Modulation of electrically evoked acetylcholine release in cultured rat septal neurones

The electrically evoked release of acetylcholine and its modulation via auto- and heteroreceptors were studied in primary cell cultures prepared from embryonic rat septum (ED 17). Cultures were grown for 1, 2 or 3 weeks on circular, poly D-lysine-coated glass coverslips. They developed a dense network of non-neuronal and neuronal cells, only some of which were immunopositive for choline acetyltransferase. To measure acetylcholine release, the cells on the coverslips were pre-incubated with [3H]choline (0.1 micromol/L), superfused with modified Krebs-Henseleit buffer at 25 degrees C and electrically stimulated twice for 2 min (S1, S2; 3 Hz, 0.5 ms, 90-100 mA). The electrically evoked overflow of [3H] from the cells consisted of approximately 80% of authentic [3H]Ach, was largely Ca2+-dependent and tetrodotoxin sensitive, and hence represents an action potential-evoked, exocytotic release of acetylcholine. Using pairs of selective agonists and antagonist added before S2, muscarinic autoreceptors, as well as inhibitory adenosine A1- and opioid mu-receptors, could be detected, whereas delta-opioid receptors were not found. Evoked [3H] overflow from cultures grown for 1 week, although Ca2+ dependent and tetrodotoxin sensitive, was insensitive to the muscarinic agonist oxotremorine, whereas the effect of oxotremorine on cells grown for 3 weeks was even more pronounced than that in 2-week-old cultures. In conclusion, similar to observations on rat septal tissue in vivo, acetylcholine release from septal cholinergic neurones grown in vitro is inhibited via muscarinic, adenosine A1 and mu-opioid receptors. This in vitro model may prove useful in the exploration of regulatory mechanisms underlying the expression of release modulating receptors on septal cholinergic neurones.