How important are Rho GTPases in neurosecretion?

Rho GTPases are small GTP binding proteins belonging to the Ras superfamily which act as molecular switches that regulate many cellular function including cell morphology, cell to cell interaction, cell migration and adhesion. In neuronal cells, Rho GTPases have been proposed to regulate neuronal development and synaptic plasticity. However, the role of Rho GTPases in neurosecretion is poorly documented. In this review, we discuss data that highlight the importance of Rho GTPases and their regulators into the control of neurotransmitter and hormone release in neurons and neuroendocrine cells, respectively.

Desensitisation of chromaffin cell nicotinic receptors does not impede catecholamine secretion during acute hypoxia in rainbow trout (Oncorhynchus mykiss).

Experiments were performed on adult rainbow trout (Oncorhynchus mykiss) in vivo using chronically cannulated fish and in situ using a perfused posterior cardinal vein preparation (i) to characterise the desensitisation of chromaffin cell nicotinic receptors and (ii) to assess the ability of fish to secrete catecholamines during acute hypoxia with or without functional nicotinic receptors. Intra-arterial injection of nicotine (6.0x10(-)(7 )mol kg(-)(1)) caused a rapid increase in plasma adrenaline and noradrenaline levels; the magnitude of this response was unaffected by an injection of nicotine given 60 min earlier. Evidence for nicotinic receptor desensitisation, however, was provided during continuous intravenous infusion of nicotine (1.3x10(-)(5 )mol kg(-)(1 )h(-)(1)) in which plasma catecholamine levels increased initially but then returned to baseline levels. To ensure that the decline in circulating catecholamine concentrations during continuous nicotine infusion was not related to changes in storage levels or altered rates of degradation/clearance, in situ posterior cardinal vein preparations were derived from fish previously experiencing 60 min of saline or nicotine infusion. Confirmation of nicotinic receptor desensitisation was provided by demonstrating that the preparations derived from nicotine-infused fish were unresponsive to nicotine (10(-)(5 )mol l(-)(1)), yet remained responsive to angiotensin II (500 pmol kg(-)(1)). The in situ experiments demonstrated that desensitisation of the nicotinic receptor occurred within 5 min of receptor stimulation and that resensitisation was established 40 min later. The ability to elevate plasma catecholamine levels during acute hypoxia (40-45 mmHg; 5.3-6.0 kPa) was not impaired in fish experiencing nicotinic receptor desensitisation. Indeed, peak plasma adrenaline levels were significantly higher in the desensitised fish during hypoxia than in controls (263+/-86 versus 69+/-26 nmol l(-)(1); means +/- s.e.m., N=6-9). Thus, the results of the present study demonstrate that activation of preganglionic sympathetic cholinergic nerve fibres and the resultant stimulation of nicotinic receptors is not the sole mechanism for eliciting catecholamine secretion during hypoxia.

Forearm norepinephrine spillover during standing, hyperinsulinemia, and hypoglycemia.

Plasma norepinephrine (NE) concentrations are a fallible index of sympathetic neural activity because circulating NE can be derived from sympathetic nerves, the adrenal medullas, or both and because of regional differences in sympathetic neural activity. We used isotope dilution measurements of systemic and forearm NE spillover rates (SNESO and FNESO, respectively) to study the sympathochromaffin system during prolonged standing, hyperinsulinemic euglycemia, and hyperinsulinemic hypoglycemia in healthy humans. Prolonged standing led to decrements in blood pressure without increments in heart rate, the pattern of incipient vasodepressor syncope. FNESO was not increased (0.58 +/- 0.20 to 0. 50 +/- 0.21 pmol. min-1. 100 ml tissue-1), suggesting that the approximately twofold increments in plasma NE and SNESO were derived from sympathetic nerves other than those in the forearm (with a possible contribution from the adrenal medullas). Hyperinsulinemia per se (euglycemia maintained) stimulated sympathetic neural activity, as evidenced by increments in FNESO (0.57 +/- 0.11 to 1.25 +/- 0.25 pmol. min-1. 100 ml tissue-1, P < 0.05), but not adrenomedullary activity. Hypoglycemia per se stimulated adrenomedullary activity (plasma epinephrine from 190 +/- 70 to 1720 +/- 320, pmol/l, P < 0.01). Although SNESO (P < 0.05) and perhaps plasma NE (P < 0.06) were raised to a greater extent during hyperinsulinemic hypoglycemia than during hyperinsulinemic euglycemia, FNESO was not. Thus these data do not provide direct support for the concept that hypoglycemia per se also stimulates sympathetic neural activity.

Frog chromaffin and adrenocortical cell co-cultures: a model for the study of medullary control of corticosteroidogenesis.

Phylogenetic, physiological and morphological evidence indicates that interactions between chromaffin and adrenocortical cells are involved in the differentiation and maintenance of function of both cell types. Chromaffin-adrenocortical interaction has become recognized as an important component of adrenocortical regulation; however, the mechanisms by which chromaffin cells modulate adrenocortical function are not well understood. To study directly chromaffin-adrenocortical cellular interactions, we developed primary frog (Rana pipiens) adrenal co-cultures. In these co-cultures, chromaffin cells extend processes that project towards or onto adrenocortical cells, mimicking their organization in vivo and indicating a potential for interaction between the two cell types. Cell survival and differentiation were optimized using a combination of NGF, FGF and histamine to enhance neurite outgrowth and fetal calf serum plus 10(-10) M ACTH to maintain steroidogenesis. Characterization of the cells by immunocytochemistry and histochemistry showed that chromaffin cells maintain expression of catecholamine biosynthetic enzymes and that adrenocortical cells maintain expression of steroidogenic enzymes. Furthermore, chromaffin cells release catecholamines upon stimulation with carbamylcholine or potassium while adrenocortical cells sustain a basal secretion rate of aldosterone and corticosterone that is augmented 10-40-fold by 0.1 nM to 10 nM ACTH. We therefore propose that these co-cultures serve as a useful model system to study the cellular and molecular mechanisms by which chromaffin cells modulate adrenocortical cell function.

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells.

Toxin-gamma (Tgamma) from the Brazilian scorpion Tityus serrulatus venom caused a concentration- and time-dependent increase in the release of norepinephrine and epinephrine from bovine adrenal medullary chromaffin cells. Tgamma was approximately 200-fold more potent than veratridine judged from EC50 values, although the maximal secretory efficacy of veratridine was 10-fold greater than that of Tgamma (1.2 vs. 12 microg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+ concentration ([Ca2+]o), when 30 microM veratridine plus 0.45 microM Tgamma were used. Tgamma (0.45 microM) doubled the basal uptake of 45Ca2+, whereas veratridine (100 microM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen when Tgamma and veratridine were combined; this was particularly pronounced at 5 mM [Ca2+]o. Veratridine induced oscillations of cytosolic Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells without elevation of basal levels. In contrast, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma with veratridine increased the shift of the I-V curve to the left, resulting in a greater recruitment of Na+ channels at more hyperpolarizing potentials. This led to enhanced and more rapid accumulation of Na+ in the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and Ca2+ entry and secretion.

Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions.

This study was carried out to characterize the set of voltage-dependent Ca2+ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM) Ba2+ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of -80 mV, averaged peak current in 10 mM Ba2+ was around 1 nA, and in 2 mM Ba2+ 0.36 nA. When using 2 mM Ba2+ as the charge carrier, nifedipine (3 microM) blocked IBa by 40-45%. omega-Conotoxin GVIA (1 microM) caused 26% inhibition, while omega-conotoxin MVIIC (3 microM) produced a 48% blockade. At low concentrations (20 nM), omega-agatoxin IVA caused 5-15% of current inhibition, while 2 microM gave rise to a 35-40% blockade. In 10 mM Ba2+, the blocking effects of nifedipine (40%) and omega-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba2+. In contrast, blockade by omega-conotoxin MVIIC was markedly reduced in 10 mM Ba2+ (20-25%) as compared to 10 mM Ba2+ (48%). The blocking actions of omega-agatoxin IVA (2 microM) were also slowed down in 10 mM Ba2+, though the final blockade was unaffected. In 2 mM Ba2+, IBa was quickly inhibited by over 94% with combined nifedipine + omega-conotoxin MVIIC + omega-conotoxin GVIA; in 10 mM Ba2+, IBa was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type (60%) high-threshold voltage-dependent Ca2+ channels. The current carried by non-L-type Ca2+ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also indicate that the fraction of current blocked by omega-conotoxin MVIIC and omega-agatoxin IVA might considerably change as a function of the Ba2+ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current is not expressed in mouse chromaffin cells.

Calcium channel current facilitation in porcine adrenal chromaffin cells.

The mechanisms of depolarizing-prepulse-induced facilitation of Ca2+ channel current were investigated in a study of porcine chromaffin cells. The Ba2+ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed "facilitation". This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased with an increase in the interpulse interval. For example, the Ba2+ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba2+ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) decreased the Ba2+ current to 0.59 times the control and GDP beta S increased it to 1.19. However, neither GTP gamma S nor guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) changed the amplitude of the Ba2+ current that was facilitated by the conditioning pulse. Thus, GTP gamma S increased the facilitation ratio to 2.05 and GDP beta S decreased it to 1.05. Furthermore, the facilitation of the Ba2+ current was abolished by omega-conotoxin GVIA but not by either omega-agatoxin IVA or nifedipine. These results suggest that, in porcine chromaffin cells, there is a omega-conotoxin GVIA-sensitive N-type Ca2+ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse.

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-[gamma-S], 100-250 microM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5-50 microM) and genistein (200 microM) 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 microM) 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 microM), phenylarsine oxide (10 microM), and ZnCl2 (500 microM), 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.

Angiotensins stimulate catecholamine release from the chromaffin tissue of the rainbow trout.

Immunohistochemical and pharmacological techniques were utilized to investigate the relationships between angiotensins and catecholamine release from the chromaffin tissue of rainbow trout (Oncorhynchus mykiss). Double labeling with [Asp1, Ile5]angiotensin II-fluorescein isothiocyanate (ANG II-FITC) and anti-dopamine beta-hydroxylase revealed specific ANG II binding sites on chromaffin cells. Injection (1 nmol/kg body wt) of either ANG II-FITC, [Asn1, Val5, Asn9]ANG I, [Asp1, Ile5, His9]ANG I, [Asn1, Val5]ANG II, [Asp1, Val5]ANG II, or [Asp1, Ile5]ANG II elicited catecholamine release from in situ perfusion preparations of the head kidney. Catecholamine release elicited by [Asn1, Val5]ANG II (10(-13) to 10(-7) mol/kg body wt) was dose dependent, and the secretion of epinephrine (Epi) was greater than that of norepinephrine (NE). Relative to the results obtained with the [Asn1, Val5]ANG II treatment (1 nmol/kg body wt), Epi release was 72 and 82% lower in response to injections (1 nmol/kg body wt) of [Asn1, Val5]ANG I [amino acid (AA) positions 1-7] and [Asn1, Val5]ANG I (AA 1-6), respectively. Pretreatment with either losartan (10(-5) M), PD-123319 (10(-5) M), or hexamethonium (10(-3) M) had no effect on [Asn1, Val5]ANG II-elicited catecholamine release. Pretreatment with captopril (10(-4) M) significantly reduced [Asn1, Val5, Asn9]ANG I-elicited Epi and NE release and decreased basal catecholamine release. These results provide direct evidence that angiotensins can elicit catecholamine release from the chromaffin tissue via specific ANG II binding sites and indicate that the synthesis of ANG II may be either local or systemic.

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells.

Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca2+ current (ICa) and exocytosis. The dependency of exocytosis on ICa evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of ICa, implying the existence of a Ca2+-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca2+ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca2+ concentration. Pertussis toxin blocked the inhibitory effects of ATP on ICa and exocytosis. Although RB-2, a P2Y antagonist, blocked the inhibitory effect of ATP on ICa, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited ICa via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of ICa by activation of a pertussis-toxin-sensitive G-protein linked to P2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis.

Somatostatin receptor regulation of gastric enterochromaffin-like cell transformation to gastric carcinoid.

BACKGROUND: Although somatostatin is recognized as an inhibitor of neuroendocrine cell secretion, its effect on cell proliferation has not been well defined. Generation of low acid and hypergastrinemia through irreversible H2-receptor blockade (loxtidine) in the African rodent mastomys results in gastric carcinoids (ECLomas) within 4 months. This study was undertaken to evaluate and characterize the precise somatostatin receptor (SSTR) subtype on the mastomys enterochromaffin-like (ECL) cell and to define its role in the regulation of ECL cell secretion and proliferation. METHODS: A pure preparation (approximately 90%) of ECL cells was derived by a combination of pronase digestion and density gradient separation. We assessed the effect of somatostatin (10(-15) to 10(-7) mol/L) on gastrin-stimulated ECL cell histamine secretion and DNA synthesis (bromodeoxyuridine uptake). SSTR2 subtype was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) using gene specific primers and mRNA isolated from normal and hypergastrinemia-induced ECLoma. The polymerase chain reaction product was confirmed by Southern analysis, subcloned, and sequenced. RESULTS: Somatostatin inhibited both gastrin-stimulated histamine secretion (IC50, 5 x 10(-13) mol/L) and DNA synthesis (IC50, 10(-10) mol/L). SSTR2 was identified in the mastomys' brain, and both normal and tumor ECL cells and comparison of the brain and ECL cell SSTR2 nucleotide sequences revealed homology of 99%. CONCLUSIONS: The SSTR2 is expressed by the mastomys' ECL cell and ECLoma. Receptor activation inhibits both ECL cell secretory and proliferative functions.

Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations.

This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of omega-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of -80 mV and depolarising pulses to 0 mV. Mean peak IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, omega-conotoxin MVIIC (3 microM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of omega-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 microM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 microM) and omega-conotoxin GVIA (1 microM), 2 microM omega-agatoxin IVA inhibited the remaining current (about 40-45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/omega-conotoxin GVIA/high omega-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of omega-agatoxin IVA and omega-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by omega-conotoxin MVIIC, the blockade by high concentrations of omega-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.

Extracellular calcium has distinct effects on fast and slow components of the depolarization-induced secretory response from chromaffin cells.

An increase in extracellular Ca2+ concentration from 0.25 to 10 mM enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K+ concentration (75 mM). The increment in extracellular Ca2+ concentration also increased the observed peak inward Ca2+ current in response to long (10-s) depolarizing pulses from a holding potential of -55 mV to +5 mV, from about -26 to -400 pA. However, the total amount of Ca2+ influx into the cell only increased when the extracellular Ca2+ concentration was raised from 0.25 to 1 mM and then remained constant up to 10 mM extracellular Ca2+. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 mM extracellular Ca2+, of approximately 4 and 41 s, being the fast component of secretion produced by the exocytosis of approximately 220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca2+ concentration.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes.

Cografting with polymer-encapsulated human nerve growth factor-secreting cells and chromaffin cell survival and behavioral recovery in hemiparkinsonian rats.

Encapsulated cell grafting is one approach for the delivery of neurotransmitters and/or neurotrophic factors to the brain. Baby hamster kidney (BHK) cells were genetically modified to secrete high levels of human nerve growth factor (hNGF). Following polymer encapsulation, these cells were implanted into the left lateral ventricle or the left striatum 1.5 mm away from striatally cografted unencapsulated adrenal medullary chromaffin cells in hemiparkinsonian rats. Although the animals receiving adrenal medulla alone or adrenal medulla with intraventricular hNGF-secreting cell grafting did not show recovery of apomorphine-induced rotational behavior, the animals receiving adrenal medulla with intrastriatal hNGF-secreting cell implants showed a significant recovery of rotational behavior 2 and 4 weeks after transplantation. Histological analysis revealed that in animals receiving adrenal medulla with intraventricular hNGF-secreting cell grafting, the number of tyrosine hydroxylase-immunoreactive (TH-IR) surviving chromaffin cells tended to be higher (approximately five to six times) than in animals receiving adrenal medulla alone; however, this increase did not reach statistical significance. In contrast, in animals receiving adrenal medullary cells together with intrastriatal hNGF-secreting cells, the number of TH-IR surviving chromaffin cells was more than 20 times higher than that in animals receiving adrenal medullary cells alone. Analysis of retrieved capsules revealed that hNGF continued to be released by encapsulated BHK-hNGF cells after 4 weeks in vivo. Moreover, histological analysis confirmed the presence of numerous viable encapsulated BHK-hNGF cells. These results indicate the potential use of intrastriatal implantation of encapsulated hNGF-secreting cells for augmenting the survival of cografted chromaffin cells as well as promoting the functional recovery of hemiparkinsonian rats. These data indicate that this approach may have potential application for treating Parkinson's disease.

Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules.

We have used membrane capacitance measurements to assay Ca2+-triggered exocytosis in single bovine adrenal chromatin cells. Brief application of phorbol ester (PMA) enhances depolarization-evoked exocytosis severalfold while actually decreasing the Ca2+ current. Ca2+ metabolism is unchanged. Three different protocols were used to show that PMA increases the size of the readily releasable pool of secretory granules. PMA treatment leads to a large increase in amplitude, but little change in the time course of the exocytic burst that results from rapid elevation of [Ca2+]i upon photolysis of DMI-Nitrophen. Thus, PKC appears to affect a late step in secretion but not the Ca2+ sensitivity of the final step.

Potassium and chloride currents in rat gastric enterochromaffin-like cells.

The gastric enterochromaffin-like (ECL) cell secretes histamine in response to secretagogues (gastrin, acetylcholine) by calcium signaling-dependent exocytosis of intracellular vacuoles containing the hormone. ECL cells were isolated from rat fundic gastric mucosa by elutriation and density-gradient centrifugation. Currents across the plasma membrane were measured using whole cell patchclamp methods. These cells had a low conductance of 0.5 nS and resting potential of -50 mV. Depolarization activated a K+ current that was blocked by Ba2+. Steady-state current in absence of K+ was due to Cl- because of the magnitude of the reversal potential and the effects of Cl- removal. Stimulation of secretion by gastrin, cholecystokinin octapeptide (CCK-8), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate activated the Cl- conductance with a time course similar to that of histamine release. Therefore the ECL cell maintains a high resting potential, largely due to K+ currents, and stimulation of secretion activates a Cl- current, perhaps deriving from the membrane of the secretory granule that fuses with the plasma membrane. The depolarization that ensues may activate the K+ current to maintain the membrane potential during exocytosis.