Search

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 ICa by 19%, ISr by 31%, and IBa 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 microM exogenous ATP reduced (about 50%) ICa, ISr and IBa 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 microM 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.

This study uses a new strategy to investigate the hypothesis that, of the various Ca2+ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca2+ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca2+ concentrations ([Ca2+]o). So, at low [Ca2+]o the K+-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60-70% by 2 microM omega-agatoxin IVA (P/Q-type Ca2+ channel blockade), by 3 microM omega-conotoxin MVIIC (N/P/Q-type Ca2+ channel blockade), or by 3 microM lubeluzole (N/P/Q-type Ca2+ channel blockade); in high [Ca2+]o these blockers inhibited the responses by only 20-35%. At 1-3 microM omega-conotoxin GVIA (N-type Ca2+ channel blockade) or 3 microM furnidipine (L-type Ca2+ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca2+]o. Combined furnidipine plus omega-conotoxin MVIIC, omega-agatoxin IVA or omega-conotoxin GVIA exhibited additive blocking effects at both Ca2+ concentrations. The results suggest that Q-type Ca2+ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca2+ that enters the cell through a Ca2+ channel located near to chromaffin vesicles will saturate the K+ secretory response at both [Ca2+]o, i.e. 0.5 mM and 5 mM. In contrast, Ca2+ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca2+]o.

1. Latrotoxin (LTX, 1-3 nM) caused a gradual increase of the spontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs-Hepes solution containing 2.5 mM Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mM) and to acetylcholine (ACh, 30 microM). 2. The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs-Hepes deprived of CaCl2) induced secretory responses that gradually reached 400-800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mM K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100-200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]o or in electroporated cells. 3. Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole-cell Ca2+ currents or basal [Ca2+]i. In fact, LTX attenuated the K(+)- or ACh-evoked increases in 45Ca2+ uptake and [Ca2+]i. 4. It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+i transients, produced by the Na(+)-Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na(+)-dependent action potentials.

Three objectives were defined when planning this study: (i) to identify binding sites for [ 125 I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 μM for 10 sec), histamine (10 μM for 10 sec) or high K + (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [ 125 I]-apamin gave a saturation curve whose Scatchard transformation produced a K d of 132 pM and a B max of 0.72 fmol/10 6 cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K + channels) did not inhibit [ 125 I]apamin binding. Binding was blocked by apamin and by d -tubocurarine, two blockers of small-conductance Ca 2+ -activated K + channels (SK channels). The number of binding sites for [ 125 I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per μm 2 surface area. Apamin (1 μM) enhanced the secretory response to histamine (10 μM), DMPP (1 or 5 μM) and high K + (20 or 35 mM) by 2–3-fold. The response to 70 mM K + , however, was unaffected. Apamin also enhanced the peak [Ca 2+ ] i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca 2+ channels, the access of Ca 2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.

Potassium-stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K+ in the presence of 2 mM Ca2+ for 10 s, applied at 5-min intervals) was inhibited by the dihydropyridine furnidipine (3 microM) by 50%. omega-Conotoxin MVIIC (CTx-MVIIC, 3 microM) also reduced the secretory response by about half. Combined CTx-MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca2+ uptake and cytosolic Ca2+ concentrations ([Ca2+]i) in K(+)-depolarized cells were partially blocked by furnidipine or CTx-MVIIC, and completely inhibited by both agents. The whole cell current through Ca2+ channels carried by Ba2+ (IBa) was partially blocked by CTx-MVIIC. Although omega-conotoxin GVIA (CTx-GVIA, 1 microM) and omega-agatoxin IVA (Aga-IVA, 0.2 microM) partially inhibited 45Ca2+ entry, IBa and the increase in [Ca2+]i, the combination of both toxins did not affect the K(+)-evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q-like Ca2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q- and L-type Ca2+ channels, but not N- or P-types are localized near exocytotic active sites in the plasmalemma.

Objetivos. Evaluar 1) la percepción materna (PM) del peso del hijo, 2) la PM de sobrepeso obesidad (SP-OB) de niños no emparentados (NNE), 3) la PM de la imagen con riesgo de salud y 4) el reconocimiento de riesgos de salud del SP-OB. Material y métodos. Participaron 325 diadas madre-hijo. Se utilizó un panel de siete imágenes. El índice de masa corporal medido del hijo fue el estándar de oro. Resultados. 84 de 100 madres de niños con SP-OB no percibieron adecuadamente el peso del hijo. El 97.5% percibió el SP-OB de NNE y 83.1% la imagen con SP-OB con riesgo de salud. La mayoría reconoció riesgos de salud. Conclusión. Es necesario diseñar programas de intervención multidisciplinarios para ayudar a las madres de niños con SP-OB a reconocer de forma adecuada el peso de su hijo y así disminuir la prevalencia de SP-OB en dicha población.

To evaluate 1) maternal perception (MP) of their child's weight, 2) MP of overweight-obesity (OW-OB) of unrelated children (URC),3) MP of image with health risk, and 4) recognition of OW-OB health risks.A total of 325 mother-child dyads participated. A panel with 7 images was used.The child's body mass index was the gold standard.A total of 84 of 100 mothers of OW-OB children did not accurately perceive their children's weight. Of the total sample, 97.5% perceived the OW-OB of URC and 83.1% the OW-OB image with health risk. Most of the mothers recognized the health risks.It is necessary to design multidisciplinary intervention programs to help mothers of OW-OB children accurately recognize their children's weight and, thus, reduce the high prevalence of OW-OB in our population.

A life-threatening intoxication, requiring ventilatory support, is reported from the ingestion of 3.05 g of buflomedil, a peripheral vasodilator. Buflomedil at a dosage of 50 mg/kg weight produces seizures. Hypotension is not a specific symptom of intoxication. Buflomedil poisoning can be easily confused with other drugs, especially tricyclic antidepressants.

This study examines the effects of a 6-week exposure to 1% NaCl in tap water with and without desoxycorticosterone acetate (DOCA) on renal alpha adrenoceptors and systolic blood pressure (SBP) in spontaneously hypertensive adult rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. On normal sodium intake, SHR had higher renal alpha 1- (p less than 0.05) and alpha 2-adrenoceptor densities (p less than 0.001) and SBP (p less than 0.001) than WKY rats. Although WKY rats given either 1% NaCl, DOCA, or DOCA plus 1% NaCl developed hypertension after 6 weeks of treatment, only 1% NaCl administration for the same period produced an increase in the alpha 1-adrenoceptor density when compared to the control (p less than 0.001). In SHR, ingestion of 1% NaCl or DOCA plus 1% NaCl increased the already elevated alpha 2-adrenoceptor density (p less than 0.001) and SBP even more in this strain after 6 weeks of treatment. Equilibrium dissociation constants (Kd), however, were similar for both classes of receptors in experimental and control rats. The results of this study indicate that only the combination of 1% NaCl in tap water and DOCA administration is effective in accelerating hypertension in adult SHR. On the other hand, in this form of genetic rat hypertension, exaggerated sodium intake with or without DOCA administration could presumably be correlated with the increased renal alpha 2 adrenoceptors observed in these animals.

The characteristics of the binding sites for the Conus magus toxins omega-conotoxin MVIIC and omega-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (IBa), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] omega-conotoxin GVIA to bovine adrenal medullary membranes was displaced by omega-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] omega-conotoxin MVIIC, which was displaced by omega-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by omega-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC50 values of around 1 nM). Both omega-conotoxin MVIIC and MVIID blocked IBa by 70-80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by omega-conotoxin MVIIC. This was not the case for omega-conotoxin MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of omega-conotoxin MVIIC did not reverse the blockade of IBa while in the case of omega-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by omega-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring IBa. Thus, 1 microM of each of omega-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 microM of each of omega-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by omega-conotoxin GVIA (1 microM). In contrast, at 1 microM both omega-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca2+ channels that contribute to the regulation of secretion and are sensitive to omega-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for omega-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.

Repetitive application of short depolarizing K+ pulses (70 mM K+, 2 mM Ca2+ Krebs-HEPES solution, for 10 s every 5 min) produced reproducible catecholamine secretory responses from superfused bovine chromaffin cells. At 10 microM for 15 min, the piperazine derivatives dotarizine, flunarizine and lidoflazine inhibited secretion by around 90%; cinnarizine halved the secretory response. Recovery of secretion after 30-min washout with Krebs-HEPES solution amounted to 75% in the case of dotarizine, 8% for flunarizine, 46% for lidoflazine and 21% for cinnarizine. The benzothiazol derivatives (10 microM) (+)-S-lubeluzole and R91154 (the (-)-R-enantiomer of lubeluzole) blocked the response by 75%; sabeluzole inhibited secretion by only 34% and R56865 (N-[1-(4-(4-fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiaz olamine) by 61%. Recoveries were around 70% in the case of these four benzothiazol derivatives. The diphenylbutyl-piperazine derivatives fluspirilene and penfluridol inhibited secretion by over 80%; no recovery was produced after 30-min washout. The inhibition of secretion was time dependent, as the recovery of the response was. Blockade of secretion by dotarizine and flunarizine occurred even in the absence of intermittent K+ stimulations of the cells. No obvious correlation was seen between the octanol/water partition coefficients of the ten compounds tested (that ranged between 6 and 4.61), the rate and extent of blockade of secretion, and the recovery of the secretory response upon washout. Rather than non-specific actions on ion channels (and secretion) due to their high lipophilicity, we believe that blockade of various Ca2+ channels relates to their binding properties to specific channel micro and macrodomains, as the case might be for 'narrow' (omega-conotoxin GVIA) and 'wide-spectrum' (omega-conotoxin MVIIC) peptide toxins.

The effects of high sodium intake (drinking 1% NaCl), DOCA and DOCA + 1%NaCl for 6 weeks on renal alpha 1- and alpha 2-adrenoceptors and on systolic blood pressure (SBP) were examined in young spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). On normal sodium intake, SHR rats had higher renal alpha 1 (p less than .001) and alpha 2-adrenoceptor densities (p less than .001) and SBP (p less than .001) than WKY rats. Although, WKY rats given either 1% NaCl, DOCA, and DOCA + 1% NaCl developed hypertension after 6 weeks of treatment, only 1% NaCl administration for the same period produced an increase in the alpha 1- and alpha 2-adrenoceptor densities when compared to the control (p less than .01 and p less than .001, respectively). In the SHR rats, to the contrary, ingestion of 1% NaCl and DOCA + 1% NaCl increased the already elevated alpha 2-adrenoceptor density (p less than .001) and SBP even more in this strain after 6 weeks of treatment. Equilibrium dissociation constants (KD), however, were similar for both classes of receptors in experimental and control rats. This study indicates that postweaning exposure of the WKY and SHR rats to a high salt treatment and DOCA can influence the renal alpha-adrenoceptor densities. Although the functional significance of the changes is unclear, it is reasonable to speculate that postweaning exposure to a hypertensinogenic stimuli such as a 1% NaCl and/or DOCA may ultimately interfere with the functional development of the kidney differently in rats genetically predisposed to hypertension (SHR) from normotensive (WKY) rats.

1. Dotarizine is a novel piperazine derivative structurally related to flunarizine that is currently being evaluated in clinical trials for its antimigraine and antivertigo effects. This clinical profile may be related to its Ca2+ antagonist properties. Therefore, the actions of both compounds as calcium antagonists were compared in bovine chromaffin cells. 2. Dotarizine and flunarizine blocked 45Ca2+ uptake into K+ depolarized chromaffin cells (70 mM K+/0.5 mM Ca2+ for 60 s) in a concentration-dependent manner, with IC50s of 4.8 and 6.7 microM, respectively. 3. Dotarizine and flunarizine also inhibited the whole-cell Ca2+ and Ba2+ currents (ICa, IBa) in voltage-clamped chromaffin cells, induced by depolarizing test pulses to 0 mV, during 50 ms, from a holding potential of -80 mV. Blockade exhibited IC50s of 4 microM for dotarizine and 2.2 microM for flunarizine. Dotarizine increased the rate of inactivation of ICa and IBa; inhibition of whole-cell currents was use-dependent. 4. Transient increases of the cytosolic Ca2+ concentration, [Ca2+]i, produced by K+ stimulation (70 mM K+ for 5 s) of single fura-2-loaded chromaffin cells, were also inhibited by dotarizine and flunarizine with IC50s of 1.2 and 0.6 microM, respectively. Upon washout of dotarizine, the [Ca2+]i increases recovered fully after 5-10 min. In contrast, the responses remained largely inhibited 10 min after washing out flunarizine. 5. Catecholamine release induced by K+ stimulation (10-s pulses of 70 mM) was inhibited by dotarizine with an IC50 of 2.6 microM and by flunarizine with an IC50 of 1.2 microM. The blocking effects of both compounds developed slowly, and was fully established after 20-30 min of superfusion. While blockade by dotarizine quickly reversed upon its washout, that of flunarizine lasted even 25 min after washing out.6. Catecholamine release from electroporated chromaffin cells triggered by 10 micro M Ca2+ was not affected by 10 micro M dotarizine or flunarizine.7. Overall, the results suggest that dotarizine behaves as a Ca2+ antagonist in bovine chromaffin cells. It exhibits a potency similar to flunarizine in blocking Ca2+ entry, Ca2+ channels, Cai2+ signals and secretion. The dotarizine effects are readily reversible suggesting that in contrast to flunarizine, it does not accumulate in cells. Dotarizine is devoid of intracellular effects on the secretory machinery. All its blocking effects on Ca2+ entry, [Ca2+]i rise and secretion are probably due to blockade of various Ca2+channel subtypes in chromaffin cells. This blockade is use-dependent and seems to be due to the increase by dotarizine of the rate of Ca2+ channel inactivation.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.