gamma-Aminobutyric acid elevates cytosolic Ca in bovine chromaffin cells

Measurements of the cytosolic Ca concentration ([Ca]i) with the Ca-sensitive dye, fura-2, showed that in intact, but not in voltage-clamped, bovine chromaffin cells gamma-aminobutyric acid (GABA, 10 microM) elicited a transient increase in [Ca]i. The Ca transient of intact cells was inhibited by bicuculline (20 microM), by removal of extracellular Ca or by treatment with the Ca channel blocker cobalt (2.5 mM), and enhanced by lowering the extracellular Cl. We conclude, that GABA elevates [Ca]i by inducing a GABAA-receptor-linked Cl current which depolarizes the cell membrane sufficiently to activate potential-operated Ca channels and cause Ca entry into the cell.     

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells

 The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl^– current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5’-0-(3-thiotriphosphate) (GTP-[γ-S], 100–250 μM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5–50 μM) and genistein (200 μM) did not inhibit the volume-sensitive Cl^– current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 μM) applied intracellularly prevented activation of the current. Voltage-activated Na⁺ and Ca²⁺ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10–100 μM), phenylarsine oxide (10 μM), and ZnCl₂ (500 μM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl^– current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl^– channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed. Received: 27 June / Received after revision: 15 September / Accepted: 16 September     

Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.     

Vesicle movements are governed by the size and dynamics of F-actin cytoskeletal structures in bovine chromaffin cells

Dense vesicles can be observed in live bovine chromaffin cells using fluorescent reflection confocal microscopy. These vesicles display a similar distribution, cytoplasmic density and average size as the chromaffin granules visualized by electron microscopy. In addition, the acidic vesicles labeled with Lysotracker Red comprised a subpopulation of the vesicles that are visualized by reflection fluorescence. A combination of fluorescence reflection and transmitted light images permitted the movements of vesicles in relation to the cortical cytoskeleton to be studied. The movement of vesicles located on the outside of this structure was restricted, with an apparent diffusion coefficient of 1.0+/-0.4 x 10(-4) microm(2)/s. In contrast, vesicles located in the interior moved much more freely and escaped from the visual confocal plane. Lysotracker labeling was more appropriate to study the movement of the faster moving vesicles, whose diffusion coefficient was five times higher. Using this type of labeling we confirmed the restriction on cortical movement and showed a clear relationship between vesicle mobility and the kinetics of cytoskeletal movement on both sides of the cortical cytoskeleton. This relationship was further emphasized by studying cytoskeletal organization and kinetics. Indeed, an estimate of the size of the cytoskeletal polygonal cages present in the cortical region and in the cell interior agreed well with the calculation of the theoretical radius of the cages imprisoning vesicle movement. Therefore, these data suggest that the structure and kinetics of the cytoskeleton governs vesicle movements in different regions of chromaffin cells.     

Excitatory and inhibitory actions of isoflurane in bovine chromaffin cells

Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing CNS function. Many anesthetics, including isoflurane, are thought to modulate and/or directly activate GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors. We previously showed that activation of the GABA(A) receptors, with specific agonists, leads to cellular excitation resulting from the depolarized anion equilibrium potential. In this study, our goal was to determine whether isoflurane mimicked this response and to explore the functional consequences of this activation. Furthermore, we sought to study the actions of isoflurane on nicotinic acetylcholine receptors (nAChRs) as they mediate the "sympathetic drive" in these cells. For these studies the Ca(2+)-indicator dye fura-2 was used to assay [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release. We show that bovine adrenal chromaffin cells were excited by isoflurane at clinically relevant concentrations. Isoflurane directly activated GABA(A) receptors found in chromaffin cells, which depolarized the cells and elevated [Ca(2+)](i). Application of isoflurane directly to the chromaffin cells elicited catecholamine secretion from these cells. At the same time, isoflurane suppressed activation of nAChRs, which presumably blocks "sympathetic drive" to the chromaffin cells. These latter results may help explain why isoflurane produces the hypotension observed clinically.     

Xenotransplantation of microencapsulated bovine chromaffin cells into hemiparkinsonian monkeys

This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs, BCCs microencapsulated in alginate-polylysine-alginate (ALA) membranes as well as empty microencapsules were grafted into the neostriatum of the hemiparkinsonian monkeys. Following the transplantation the hemiparkinsonian symptoms subsided and the number of rotations induced by apomorphine decreased for up to nine months in the group of recipients grafted with microencapsulated BCCs, while only a temporary improvement (one month) was detected in the recipients of the unencapsulated BCCs. No change was observed in the recipients of empty microencapsules. Dopamine and its metabolites were found considerably depleted in the MPTP-lesioned side versus the unlesioned side of the neostriatum in the hemiparkinsonian monkeys(P<0.05).     

Temperature dependence of release of vesicular content in bovine chromaffin cells

Recent studies suggest that the time course of secretion of the vesicular content in bovine chromaffin cells is much slower than in the peripheral or the central nervous system, but the reasons for this marked difference are not known. In this study we try to assess the importance of factors that may influence the time course of release of the vesicular content of bovine chromaffin cells, namely: (1) diffusion of catecholamines in the extracellular solution, (2) dissociation of catecholamines from the matrix of chromogranin A, and (3) the kinetics of opening and closing of the fusion pore. The temperature dependence of the time course and the amplitude of the spontaneous current spikes were examined using the carbon filament recording technique in amperometric mode. The change in amplitude was not statistically significant, but both the rise and the decay times were shortened (from 29 ± 12 to 16 ± 5 ms, and from 87 ± 26 to 57 ± 11 ms respectively) as temperature was raised by 20 °C [from 15 to 35 °C; n = 6; the changes were statistically significant at the level of P = 0.05; their respective temperature coefficients (Q ₁₀) were 1.4 and 1.3]. The areas underneath the spontaneous current spikes, however, were not altered significantly. Neither the relationship between the rise and the decay times nor the frequency of occurrence of the spontaneous current spikes changed consistently as the temperature was raised. However, the frequency histograms could, in all cases, be well described by a monoexponential function. It is concluded that the release of catecholamine content from the individual vesicles in bovine chromaffin cells is probably mostly determined by the dissociation of catecholamines from the matrix of chromogranin A. Received: 10 October 1995/Received after revision and accepted: 29 April 1996     

Host-graft relationships of isolated bovine chromaffin cells in rat periaqueductal grey

Summary The transplantation of chromaffin cells from the adrenal medulla into pain modulatory regions of the CNS has previously been shown to reduce pain sensitivity, most likely via local release of neuroactive substances from the transplanted cells. The ready availability of bovine adrenal glands, as well as the high levels of opioid peptides produced by their chromaffin cells, make these glands a potentially valuable donor source for antinociception studies. However, the success of these xenografts depends on their ability to survive and integrate within the host CNS.The aim of the present study was to assess host-graft relationships of bovine chromaffin cells transplanted to the rat CNS. We have found that isolated bovine chromaffin cells survive for at least three months in the rat periaqueductal grey, with no evidence of immunological response following a short-term course of immunosuppressant treatment. In the early stages following transplantation, only minor pathology is found at the injection site, which apparently recovers completely at later stages. The host-graft borders are not well demarcated, in contrast to solid tissue grafts.Neuronal processes of host origin, forming numerous synapses with the transplanted bovine chromaffin cells, are apparent by three weeks following transplantation. Migration also occurs from the graft into the host parenchyma, as evidenced by individual chromaffin cells found near host parenchymal blood vessels. The clusters of chromaffin cells found in the graft itself are generally not very vascular, in contrast to solid tissue grafts. The chromaffin cell clusters are surrounded by blood vessels of the non-fenestrated CNS type at the host-graft border. It is likely that the small size of the graft does not require extensive angiogenesis. The lack of fenestrated peripheral-type endothelial capillaries, normally seen in adrenal medullary tissue grafts, may contribute to the survival of these xenografts in the rat brain.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

经冻存的APA微囊牛肾上腺髓质细胞功能研究

目的观察海藻酸钠-聚赖氨酸-海藻酸钠(alginate-polylysine-alginate,APA)微囊牛肾上腺髓质细胞经液氮冻存前后的微囊形态、细胞活性和儿茶酚胺分泌功能变化。方法APA微囊牛肾上腺髓质细胞(牛嗜铬细胞,bevine chromaffin cell,BCC)的冻存以DMSO为保护剂,冻存采取循序缓慢降温,复苏采取快速复温。通过微囊形态、细胞活性和儿茶酚胺分泌量检测观察细胞功能变化。结果与冻存前相比,冻存复苏后的APA微囊BCC形态及细胞活性无明显变化,儿茶酚胺分泌量约为冻存前的80%。结论经冻存复苏的APA微囊BCC仍然保持了较好的形态、细胞活性和儿茶酚胺分泌功能。     

Small-conductance Ca2+-activated K+ channels in bovine chromaffin cells

Simultaneous whole-cell patch-clamp and fura-2 fluorescence [Ca²⁺]_i measurements were used to characterize Ca²⁺-activated K⁺ currents in cultured bovine chromaffin cells. Extracellular application of histamine (10 M) induced a rise of [Ca²⁺]_i concomitantly with an outward current at holding potentials positive to –80 mV. The activation of the current reflected an increase in conductance, which did not depend on membrane potential in the range –80 mV to –40 mV. Increasing the extracellular K⁺ concentration to 20 mM at the holding potential of –78 mV was associated with inwardly directed currents during the [Ca²⁺]_i elevations induced either by histamine (10 M) or short voltage-clamp depolarizations. The current reversal potential was close to the K⁺ equilibrium potential, being a function of external K⁺ concentration. Current fluctuation analysis suggested a unit conductance of 3–5 pS for the channel that underlies this K⁺ current. The current could be blocked by apamin (1 M). Whole-cell current-clamp recordings snowed that histamine (10 M) application caused a transient hyperpolarization, which evolved in parallel with the [Ca²⁺]_i changes. It is proposed that a small-conductance Ca²⁺-activated K⁺ channel is present in the membrane of bovine chromaffin cells and may be involved in regulating catecholamine secretion by the adrenal glands of various species.     

Molecular mechanisms of glutamate release by bovine chromaffin cells in primary culture

Previous work indicated that glutamate could be involved in the regulation of catecholamine secretion in bovine chromaffin cells. Thus, the question arises on the source of this putative regulatory glutamate. In this work we have examined the possibility that glutamate could be released from chromaffin cells. Data from this study indicate that chromaffin cells are able to release glutamate when they are stimulated by different depolarising agents such as 60 mM KCl, 1 mM 4-aminopyridine or 50 microM veratridine. The amount of glutamate released by these compounds was 0.32 nmol/10(6) cells (9.24% of cellular glutamate content), 0.275 (7.86%) and 0.158 (4.52%) for KCl, 4-AP and veratridine stimulation, respectively. All these catecholamine-secretagogues induced glutamate secretion by two mechanisms: 1) a Ca(2+)-dependent, probably exocytotic, mechanism and 2) a Ca(2+)-independent mechanism mediated by reversion of the electrogenic glutamate transporter. Analysis of Ca(2+)-dependent and independent releases for different compounds carried out by several experimental approaches, indicate that Ca(2+)-dependent release was the predominant mechanism for release induced by 4-aminopyridine (84% of total release) and high KCl (63%) whilst Ca(2+)-independent release was predominant for veratridine (67%). The Ca(2+)-dependent glutamate release evoked by depolarisation of chromaffin cells with high KCl and 4-AP could be split into both a fast and a slow kinetic component, which might correspond to the release of docked and mobilised chromaffin granules, respectively. On the other hand, depolarisation of cells with veratridine result in glutamate release with only the fast kinetic component. In the case of 60 mM KCl-evoked glutamate release, the fast component exhibited a decay time of <1 s and accounted for 0.63 nmol glu/6x10(6) cells (70% of total exocytotic release), whereas the slow component, which exhibited a decay time of 231 s, accounted for the release of 0.27 nmol glu/6x10(6) cells (30% of total exocytotic release). By contrast in the case of 4-aminopyridine the fast component of exocytosis only represents a 19% of total secretion and the slow a 81% with a decay time of 94 s. These data are very similar to those found in neurones and support the possible intracellular origin of glutamate having a role in the regulation of catecholamine secretion from chromaffin cells. In support of this, we have found that glutamate secretion could be evoked by stimulation of the nicotinic cholinergic receptors.     

The blocking effects of omega-conotoxin on Ca current in bovine chromaffin cells

The effects of omega-conotoxin (omega-CgTX) on voltage-sensitive Ca currents (ICa) were studied in cultured bovine adrenal chromaffin cells. A maximal block of ICa of 40-50% was obtained with omega-CgTX in the microM range, and was independent of the holding potential. The onset of block was both concentration- and time-dependent. In bovine chromaffin cells, Ca channels, both sensitive and insensitive to omega-CgTX, appear to be present.     

Stage-specific alteration of nucleoside membrane permeability and nitrobenzylthioinosine insensitivity in Plasmodium falciparum infected erythrocytes

In human erythrocytes, the intracellular presence of malarial parasites (Plasmodium falciparum) markedly changed the permeation characteristics of the nucleosides, adenosine and tubercidin, an adenosine analogue. We report parasite-induced changes in the kinetics of cellular uptake of the nucleosides and in the appearance in infected cells of a nucleoside permeation route of low sensitivity to the classical inhibitor of erythrocytic nucleoside transport, nitrobenzylthioinosine (NBMPR). These changes and a diminution in NBMPR effectiveness during parasite maturation to the trophozoite or schizont stage, suggest the presence in the infected cells of an altered or new nucleoside permeation mechanism of low sensitivity to NBMPR. The incorporation of adenosine into polynucleotides was also of low sensitivity to 10 microM NBMPR. Binding studies of [3H]NBMPR with both normal erythrocytes and those harbouring parasites at each morphological stage indicated that fewer high affinity NBMPR binding sites were present on cells containing mature parasites than on the uninfected cells. The apparent low sensitivity to NBMPR of nucleoside permeation in erythrocytes containing P. falciparum forms may enable therapeutic measures with cytotoxic nucleosides to be directed with selectivity toward parasite-containing cells.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannicacid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane. Our results provide the first ultrastructural demonstration of exocytosis in bovine chromaffin cells with the aid of transmission electron microscopy. It is also clear that exocytosis is the mechanism of release in both intact and permeabilized cells.     

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At –70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.