Qualitative measurements of cytosolic calcium ion concentration within isolated guinea pig nerve endings using entrapped arsenazo III

If arsenazo III is present during homogenization of brain this metallochromic indicator is entrapped within subsequently isolated synaptosomes. A large proportion of the entrapped indicator is released upon addition of digitonin to disrupt the synaptosomal plasma membrane. A similar proportion of [3H]sucrose is also trapped within synaptosomes if present in the homogenization medium, suggesting that homogenization causes a transient opening of the nerve ending as it is chopped off from the axon. Addition of the ionophore A23187 or depolarization of the plasma membrane by adding veratridine, gramicidin or increasing external K+ changes the absorbance of the entrapped dye, with peaks of absorbance around 600 and 650 nm, typical of the arsenazo III-Ca2+ complex. The response to veratridine is inhibited by the Ca2+-channel antagonist, verapamil, while that of A23187 is unaffected. The present method provides a sensitive technique for measurements of changes in cytosolic calcium ion concentrations within nerve endings.     

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.     

GABA immunoreactivity of calyceal nerve endings in the vestibular system of the guinea pig

Summary Neurotransmitters involved in the vestibular system are largely uncharacterized. On the basis of results of earlier electrophysiological and immunohistochemical experiments, glutamate and gamma-amino-butyric acid (GABA) have been proposed in both mammalian and non-mammalian species as afferent transmitters between the sensory cell and the afferent dendrite. GABA is also suspected to act as an efferent neurotransmitter in the cochlea. We describe in this study the immunocytochemical localization of GABA within the vestibular end organs in the guinea pig. GABA immunoreactivity was found in the calyceal nerve endings surrounding type I hair cells of the vestibular epithelia. The most significant labelings were obtained in the crista ampullaris. Labeling was more difficult to observe in the utricular and saccular macula. These results contribute to the recent proposal that the calyx has a secretory function, and suggest that GABA may have a modulatory influence upon the type I hair cells.     

Density distribution of guinea pig myenteric plexus nerve endings containing immunoreactive substance P

The present study was performed to investigate how myenteric plexus nerve endings containing substance P are distributed in sucrose density gradients in relation to nerve endings capable of taking up 3H-acetylcholine or 14C-noradrenaline. The peak of substance P-immunoreactivity (ISP) was found at a density of 1.157 +/- 0.001 g X ml-1, that of 3H-radioactivity at 1.160 +/- 0.002 and that of 14C-radioactivity at 1.162 +/- 0.002 g X ml-1 (mean +/- SEM, N = 6); there was considerable overlap. In a second set of experiments, the resuspended P2-pellet was layered upon a discontinuous density gradient consisting of 0.6, 1.0, 1.2 and 1.4 M sucrose. Nine fractions were recovered. There was a 2.5-3.4-fold increase in the relative specific activity of ISP in the 1.2 M fraction (density = 1.154 g X ml-1) and the adjoining interfaces. Conventional electron microscopy showed that synaptosomal elements were present in the transmitter-enriched fractions. It is concluded that the substance P-containing nerve endings of the guinea pig myenteric plexus co-distribute (and may be co-purified with) nerve endings utilizing noradrenaline or acetylcholine on sucrose density gradients.     

神经节内板状末梢是豚鼠食道迷走传入神经末梢的机械敏感性受体

电生理学研究发现迷走传入神经在胃肠道的特有结构——神经节内板状末梢(intraganglionic laminar endings,IGLEs)具有感受机械刺激的功能,推断其为迷走神经机械敏感性受体。但是电生理学方法不能将IGLEs的特异结构与其感受机械刺激的功能同时显示出来,而且IGLEs作为机械敏感性受体,其传导机械刺激的机制尚不清楚。本研究应用活性依赖性荧光染料 FM1-43结合牵拉刺激豚鼠食道显示激活的IGLEs结构,以期观察IGLEs是否对机械刺激敏感。同时用多种药物阻断或促进豚鼠食道IGLEs的激活以探讨IGLEs传导机械刺激的机制。应用神经顺行标记技术以验证FM1-43显示的特异结构是否为IGLEs。结果表明,牵拉刺激结合FM1-43染色显示的结构与神经顺行标记法一致,牵拉刺激组激活的IGLEs数目明显多于未牵拉组 [(90．4±9．5)％vs(10．7±2．1)％,P<0．05]。IGLEs对牵拉刺激的敏感性,表明IGLEs是迷走传入神经在胃肠道内感受机械刺激的受体。TTX,阿托品和钙离子对牵拉刺激激活IGLEs无明显影响,表明IGLEs对机械刺激的传导不需要神经递质以及动作电位的传导,而是直接通过机械门控离子通道实现的。多种TRP通道阻断剂包括SKF,gadolinium对IGLEs的激活无影响,而上皮钠离子通道阻断剂benzamil可以明显阻断IGLEs的激活,因此推断,IGLEs结构中传导机械刺激的离子通道可能属于上皮钠离子通道家族而非电压门控钠离子通道或TRP通道。     

Decreased calcium accumulation in isolated nerve endings during hibernation in ground squirrels

Resting and depolarization-induced⁴⁵CaCl₂ accumulation was compared for synaptosomes isolated from hibernating and nonhibernating ground squirrels. Channel subtype antagonists were used to identify the active voltage-sensitive calcium channel subtypes in these preparations. There was significantly less⁴⁵Ca²⁺ accumulation in synaptosomes isolated from hibernating as compared to cold-adapted nonhibernating ground squirrels in both basal (p<0.005) and depolarizing (p<0.03) media over a 30 sec to 5 min incubation period. The elevation in⁴⁵Ca²⁺ accumulation triggered by K⁺ depolarization was blocked by 50 μM CdCl₂, 1 μM ω-conotoxin MVIIC or 1 μM ω-agatoxin IVA. Inhibition was not observed with 1 μM nifedipine or with 1 μM ω-conotoxin GVIA. These results suggest that hibernation is associated with reduced presynaptic⁴⁵Ca²⁺ conductance via voltage-sensitive channels with a pharmacological sensitivity that is different from the established L-, N-, and P-types in other systems but share features of the recently described Q-type calcium channel. This decrease may reflect a cellular adaptation that helps confer tolerance to the near total cerebral ischemia associated with hibernation.     

Modeling the calcium sequestration system in isolated guinea pig cardiac mitochondria

Under high Ca²⁺ load conditions, Ca²⁺ concentrations in the extra-mitochondrial and mitochondrial compartments do not display reciprocal dynamics. This is due to a paradoxical increase in the mitochondrial Ca²⁺ buffering power as the Ca²⁺ load increases. Here we develop and characterize a mechanism of the mitochondrial Ca²⁺ sequestration system using an experimental data set from isolated guinea pig cardiac mitochondria. The proposed mechanism elucidates this phenomenon and others in a mathematical framework and is integrated into a previously corroborated model of oxidative phosphorylation including the Na⁺/Ca²⁺ cycle. The integrated model reproduces the Ca²⁺ dynamics observed in both compartments of the isolated mitochondria respiring on pyruvate after a bolus of CaCl₂ followed by ruthenium red and a bolus of NaCl. The model reveals why changes in mitochondrial Ca²⁺ concentration of Ca²⁺ loaded mitochondria appear significantly mitigated relative to the corresponding extra-mitochondrial Ca²⁺ concentration changes after Ca²⁺ efflux is initiated. The integrated model was corroborated by simulating the set-point phenomenon. The computational results support the conclusion that the Ca²⁺ sequestration system is composed of at least two classes of Ca²⁺ buffers. The first class represents prototypical Ca²⁺ buffering, and the second class encompasses the complex binding events associated with the formation of amorphous calcium phosphate. With the Ca²⁺ sequestration system in mitochondria more precisely defined, computer simulations can aid in the development of innovative therapeutics aimed at addressing the myriad of complications that arise due to mitochondrial Ca²⁺ overload.     

Dependence of the red blood cell calcium pump on the membrane potential

(1) It is shown that the rate of calcium extrusion from intact human red cells is faster at a membrane potential of approximately +50 mV (inside) than at approximately -50 mV. (2) The positive potential applied was the chloride potential of KCl cells in a K-gluconate medium when the Ca2+ sensitive K+ channel was blocked by 0.3mM quinidine. The negative potential resulted from the high K+ permeability in Ca2+ loaded cells (the cells were loaded to a Ca2+ activity in the cell water of about 50 microM). (3) It is further demonstrated that the Ca2+ affinity of the pump ATPase is decreased both at the internal (high affinity) and external (low affinity) site by increasing the proton concentration. Acidification thus inhibits internally and stimulates externally. (4) An indirect effect of the membrane potential on the pump activity via the accompanying pH shifts on either side of the membrane could be ruled out by choosing Ca2+ concentrations which are fully activating at the internal Ca2+ binding site at pH 6.5 and not yet inhibitory at the external Ca2+ binding site at pH 8. (5) The result is compatible with the assumption that the human red cell Ca-pump is exchanging Ca2+ for protons, yet is electrogenic by virtue of a stoichiometry of 1H+:1Ca2+ for this exchange.     

Dependence of the intensity of growth processes on the potential difference at the cytoplasmic membrane

The growth of organisms is a dynamic process of interrelated fluxes of substances and energy which most often proceeds in the steady-state conditions, the energy-dependent process being predominant. The rate of growth is directly proportional to the potential difference at the plasma membrane, created essentially by a passive influx of energy substance to the cell oxidative system which is a limiting link of its energization. The formation of potential difference is influenced by concentrations of biomass and energy substrate at the plasma membrane, as well as the values of electric capacity of the membrane and rate constants for the electric charge formation and expenditure.     

Modulation of the membrane potential of intact guinea pig aortic endothelium

The role of different ion conductances in regulation of the membrane potential (MP) of resting and agonist-stimulated intact endothelium from the guinea pig aorta was investigated. Under resting conditions, the MP measured by the patch-clamp technique varied within the range from –29 to –56 mV (the mean value of –40.8 ± 8.1 mV). Blockers of anomalous (inward) rectifier potassium channels cesium (100 µM) and barium (100 µM) exerted no effect on the MP of endothelium. Superfusion of preparations with calcium-free solution and application of 2 mM nickel depolarized the endothelium. ATP (10 µM) induced hyperpolarization of endothelium with the mean amplitude of 11.4 ± 0.6 mV. The initial phase of this hyperpolarization depended on the external potassium concentration and on the state of intracellular calcium stores, whereas the prolonged phase required the presence of external calcium. In the absence of external calcium, in 25% of recordings transient hyperpolarization was followed by depolarization, which was not observed after substitution of external NaCl for choline. It was concluded that basal activity of calcium-dependent potassium channels contributes to the regulation of the MP of resting endothelium. Stimulation with ATP led to activation of calcium-dependent potassium and nonselective cationic channels. Activation of the former channels produced the initial phase of hyperpolarization, whereas activation of the second channel type evoked the prolonged phase of hyperpolarization.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 260–266, November–December, 1996.     

Effects of guinea pig vasoactive intestinal peptide on the isolated perfused guinea pig heart

The pharmacological effects of guinea pig vasoactive intestinal peptide (VIP) were studied in isolated perfused guinea pig hearts. Bolus injections of VIP produced a dose-dependent tachycardia that was not affected by atenolol. A decrease in amplitude of ventricular contractions occurred in response to all doses of VIP. This response was preceded by a small increase in amplitude in 3 of 6 hearts at the highest dose. VIP produced a decrease in perfusion pressure which was prominent after coronary tone was elevated with [Arg8]-vasopressin. The present findings support speculation that VIP may have a role in the regulation of heart rate and coronary blood flow.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Effect of acidosis on the membrane potential of intact guinea pig aortic endothelial cells

The effects of a decrease in the extracellular pH from 7.4 to 6.9 on the membrane potential (MP) of intact non-stimulated guinea pig aortic endothelial cells and their ATP-induced electrical responses were studied using a whole-cell mode of the patch-clamp technique. Superfusion of the strip with CO₂-−HCO ₃ ⁻ -buffered acidic solution evoked endothelial depolarization of 6.1±1.0 mV. In Ca²⁺-free medium, after the MP had been stabilized at a depolarized value, there was no shift in the MP due to extracellular acidification to pH 6.9. In the case of using tris-buffered solution, the same drop in the extracellular pH in Ca²⁺-containing medium induced no change in the endothelial MP. Subsequent superfusion with CO₂−HCO ₃ ⁻ -buffered solution with pH 6.9 evoked endothelial depolarization of 7.3±1.5 mV. Changing from tris-buffered to CO₂−HCO ₃ ⁻ -buffered solution at a constant buffer pH 7.4 also induced endothelial depolarization, suggesting that intracellular pH is a possible factor that modulates leak Ca entry. The duration of ATP-induced endothelial hyperpolarization at pH 6.9 significantly dropped (76±5 sec, on average) compared with that at pH 7.4 (121±14 sec). It is concluded that modulatory effect of acidosis on the MP of endothelial cells and their ATP-induced electrical responses are caused by inhibition of leak and ATP-stimulated calcium entry into these cells.     

The "vesicle in a basket". A morphological study of the coated vesicle isolated from the nerve endings of the guinea pig brain, with special reference to the mechanism of membrane movements

Five points are discussed regarding the vesicular structure isolated by fractionation techniques from the brain and liver of the guinea pig. 1. One type of vesicle, fixed by OsO₄ and shown in thin sections, is identified with the coated vesicle that has been observed in many varieties of tissues. 2. The vesicle contained in a spherical polygonal "basketwork" shown by the negative-staining techniques is identical with the coated vesicle shown in sections. 3. Despite minute observation of this basketwork we could not confirm the existence of "hairlike projections" extending from the convex cytoplasmic surface of the vesicle. We are inclined to believe, therefore, that the hairlike projections are actually the superimposed visual images of the regular hexagons and pentagons of the network composing the basketwork. 4. We repeat the hypothesis originally advanced by Roth and Porter (1) that the "coating" of the coated vesicle plays a role in the mechanism of the infolding and fission of the membrane; we suggest that these events are caused by the transformation of the regular hexagons (of the coating) into regular pentagons. 5. Finally, we make a suggestion as to the nature of those vesicles which have on their surface subparticles which look like "elementary particles (2)."     

Sodium inhibits hormone release and stimulates calcium efflux from isolated nerve endings of the rat neurohypophysis

1. We studied the effects of extracellular sodium on the secretion of vasopressin (VP) and oxytocin (OT) and the efflux of 45Ca from isolated, perfused nerve endings of the rat neurohypophysis (neurosecretosomes). 2. Upon removal of sodium from the perfusing medium, basal release of VP and OT increased by 3.95 +/- 0.23- and 3.71 +/- 0.22-fold, respectively, followed by a decline to about double the levels in normal (150 mM) sodium (P less than or equal to 0.1). 3. Compared to neurosecretosomes perfused in normal (150 mM) sodium, omission of sodium from the medium augmented ionomycin-induced VP and OT secretion by 66 +/- 5- and 20 +/- 3-fold, respectively, and A23187-induced secretion was increased 1.3 +/- 0.4- and 1.3 +/- 0.1-fold (P less than or equal to 0.01 for both ionophores). 4. The inhibition of ionomycin-induced secretion by sodium was concentration dependent (P less than or equal to 0.01 for sodium greater than or equal to 5 mM); the IC50 was about 10 mM sodium for both hormones, and the Hill slope was close to -1. 5. The rate of 45Ca efflux from neurosecretosomes showed 2.7 +/- 0.1-fold stimulation upon increasing sodium from 4.5 to 150 mM (P less than or equal to 0.01). 6. Our results suggest that sodium inhibits basal and stimulated secretion at the nerve terminal, possibly by reducing intraterminal calcium through sodium/calcium exchange.     

ATP-dependent calcium sequestration and calcium/ATP stoichiometry in isolated microsomes from guinea pig parotid glands

ATP-dependent calcium uptake was studied in isolated guinea pig parotid gland microsomes. The apparent Km for free Ca2+ was 0.41, microM, the apparent Km for ATP X Mg2- 0.23 mM. The pH optimum was 6.8-7.0. Subfractionation of the microsomes revealed that the highest specific uptake activity resided in a rather dense fraction of the endoplasmic reticulum. The calcium uptake/ATPase stoichiometry was determined in the absence of exogenous magnesium in the submicrosomal fractions. It ranged from 1-2. It is concluded that in vivo the stoichiometry is the same as in sarcoplasmic reticulum, namely 2.     

Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.