Biomedical subjects
Publications and source records attributed to R M Santos.
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An uncommon case of high blood pressure secondary to subisthmic aortic coarctation and cured surgically by arterial revascularization is reported. It is probably a congenital coarctation, because the extensive clinical evaluation and condition of the patient ruled out an acquired disease of the aorta, confirmed by the morphologic operative findings and pathological examinations of the aortic wall. In this case, the clinical diagnosis was initially suggested due to an abdominal systolic-diastolic bruit, associated to hypertension and weak femoral pulses. Although doppler ultrasonography and nuclear magnetic resonance were useful diagnostic tools, a complete angiography should be performed because it is currently the most informative technique to evaluate the aorta and its collaterals, frequently involved in association with subisthmic aortic coarctation. Surgical exploration revealed a hypoplastic descending thoracic aorta and histological data confirmed the nonexistence of acquired pathology of the aortic wall. Surgical management was accomplished successfully and the patient was asymptomatic 41 months after the operation with normal blood pressure, no treatment, and a normal pattern of life. Based on this presentation, the authors describe the etiology, morphology and anatomic classification, clinical manifestations, diagnosis and management of subisthmic aortic coarctation.
We report a highly sensitive electrochemical approach suitable for the real time measurement of insulin release from single islets of Langerhans, the functional endocrine units in the pancreas. The method is based on the detection of the insulin surrogate 5-hydroxytryptamine (5-HT) by carbon fibre microelectrodes implanted in the islets. Based on the combination of this novel approach with the simultaneous microfluorometric recording of cytosolic free Ca2+ concentration ([Ca2+]i), we demonstrate that glucose-stimulated islets secrete 5-HT/insulin in a pulsatile fashion under physiological conditions, and that this activity is encoded by synchronous [Ca2+]i oscillations. The sensitivity to detect variations in minute amounts of secreted materials is partially conferred by the fact that the tracer is released into a relatively confined space (the intraislet interstitial space).
Glucose stimulation raises the pHi of pancreatic beta-cells, but the underlying mechanisms are not well understood. We have now investigated the acute effects of metabolizable (glucose and the mitochondrial substrate alpha-ketoisocaproic acid, KIC) and nonmetabolizable (high K+ and the K-ATP channel blocker tolbutamide) insulin secretagogues on the pHi of pancreatic beta-cells isolated from normal mice, as assessed by BCECF fluorescence from single cells or islets in the presence of external bicarbonate. The typical acute effect of glucose (22-30 mM) on the pHi was a fast alkalinization of approximately 0.11 unit, followed by a slower acidification. The relative expression of the alkalinizing and acidifying components was variable, with some cells and islets displaying a predominant alkalinization, others a predominant acidification, and others yet a mixed combination of the two. The initial alkalinization preceded the [Ca2+]i rise associated with the activation of voltage-sensitive Ca2+ channels. There was a significant overlap between the glucose-evoked [Ca2+]i rise and the development of the secondary acidification. Depolarization with 30 mM K+ and tolbutamide evoked pronounced [Ca2+]i rises and concomitant cytosolic acidifications. Blocking glucose-induced Ca2+ influx (with 0 Ca2+, nifedipine, or the K-ATP channel agonist diazoxide) suppressed the secondary acidification while having variable effects (potentiation or slight attenuation) on the initial alkalinization. KIC exerted glucose-like effects on the pHi and [Ca2+]i, but the amplitude of the initial alkalinization was about twice as large for KIC relative to glucose. It is concluded that the acute effect of glucose on the pHi of pancreatic beta-cells is biphasic. While the initial cytosolic alkalinization is an immediate consequence of the activation of H+-consuming metabolic steps in the mitochondria, the secondary acidification appears to originate from enhanced Ca2+ turnover in the cytoplasm. The degree of coupling between glucose metabolism and Ca2+ influx as well as the relative efficacies of these processes determines whether the acute pHi response of a beta-cell (or of a tightly coupled multicellular system such as an islet of Langerhans) is predominantly an alkalinization, an acidification, or a mixed proportion of the two.
We have investigated the effects of the phorbol ester 12-myristate 13-acetate (PMA) on depolarization-evoked Ca2+ influx and catecholamine secretion in bovine adrenal chromaffin cells. PMA (100 nM) strongly inhibited K(+)-evoked [Ca2+]i transients and Mn2+ quenching of fura-2 fluorescence. In contrast, 4 alpha-phorbol 12,13-didecanoate, a phorbol ester inactive on protein kinase C (PKC), had no effect. Maximal PMA-mediated inhibition occurred at 5-10 min incubations and were variable from cell to cell, ranging from 25 to 65% of controls. The [Ca2+]i transients evoked by the L-type Ca2+ channel activator Bay K 8644 were strongly inhibited by 100 nM PMA. PMA (0.1-10 microM) inhibited K(+)-evoked adrenaline and noradrenaline release by 23-44%. The data indicate that phorbol ester-mediated activation of PKC inhibits voltage-sensitive Ca2+ channels in chromaffin cells, leading to a prominent depression of depolarization-evoked catecholamine secretion.
A stepwise increase in extracellular Ca2+ concentration ([Ca2+]o) can evoke insulin release from pancreatic islets in the absence of secretagogues. We have investigated the ionic mechanism underlying this secretory response by recording intracellular free Ca2+ concentration ([Ca2+]i) from single mouse islets of Langerhans using ratiometric fura-2 microfluorometry. In the presence of 11 mM glucose, the [Ca2+]i undergoes fast oscillations associated with bursting electrical activity. Nifedipine (10 microM) suppressed these oscillations and markedly lowered the [Ca2+]i. Raising the [Ca2+]o from 2.56 to 12.8 mM in the continued presence of 11 mM glucose and nifedipine evoked pronounced [Ca2+]i rises of variable amplitude and time course. This effect was dose-dependent (EC50 = 3.6 mM) and remained essentially unchanged in the absence of glucose or in the presence of 3 mM glucose and nifedipine, conditions where beta-cells are hyperpolarized by approximately -25 mV. Depleting the acetylcholine-mobilizable internal Ca2+ pools by repetitively challenging the islets with acetylcholine in the absence of Ca2+ actually potentiated the standard high Ca2+ responses. The latter were strongly reduced by millimolar concentrations of Ni2+ (70% reduction at 3 mM) and by diphenylamine-2-carboxylate (DPC; IC50 = 145 microM), a blocker of nonselective cation channels. The standard high Ca2+ responses were relatively insensitive to the glycolytic inhibitor mannoheptulose. It is proposed that the high Ca(2+)-evoked [Ca2+]i responses are primarily accounted for by Ca2+ influx through dihydropyridine- and voltage-insensitive, nonselective cation channels. These channels do not appear to be under the control of glucose metabolism. Although their function is unknown, they may be essential to supplying the beta-cells with Ca2+ in the absence of stimulatory levels of fuel secretagogues.
The study of the influence of intracellular pH (pHi) changes on the mechanism underlying pancreatic beta-cell bursting has been hampered by concomitant effects on the activity of background ATP-dependent K+ (K-ATP) channels. beta-cells were made to burst in the absence of active K-ATP channels by raising external Ca2+ in the presence of 11 mM glucose and tolbutamide. An alkalinizing pHi shift (exposure to 20 mM NH4Cl) increased the burst active phase duration. Conversely, an acidifying shift (NH4Cl withdrawal) suppressed the electrical activity. This is the mirror image of the effects recorded in the absence of tolbutamide. Glibenclamide and quinine suppressed the alkalinization-evoked hyperpolarization. This study emphasizes the differential sensitivity of different beta-cell ion channels to pHi and the prevalent role of K-ATP channels as electrical transducers of cytoplasmic pH changes under regular physiological conditions.
There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by omega-conotoxin-sensitive and -insensitive Ca2+ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 microM) and neomycin (0.5 mM) reduced high K+ (50 mM)-evoked intracellular Ca2+ concentration ([Ca2+]i) transients to a larger extent than each drug separately. [Ca2+]i was measured using the fluorescent intracellular Ca2+ indicator fura-2. Neomycin (0.05-2 mM) reduced high K(+)-evoked 45Ca2+ uptake in a dose-dependent manner (IC50 = 0.09 mM). In the presence of nitrendipine (1 microM), the minimal neomycin concentration necessary for total blockade of 45Ca2+ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the 45Ca2+ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05-2 mM) inhibited the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05-2 mM) blocked high K(+)-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC50 = 0.8-1.1 mM), the blockade efficiency being enhanced in the presence of 1 microM nitrendipine (IC50 = 0.17-0.19 mM). It is concluded that neomycin (< or = 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca2+ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca2+ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.
In glucose-stimulated pancreatic beta-cells, the membrane potential alternates between a hyperpolarized silent phase and a depolarized phase with Ca2+ action potentials. The molecular and ionic mechanisms underlying these bursts of electrical activity remain unknown. We have observed that 10.2-12.8 mM Ca2+, 1 microM Bay K 8644 and 2 mM tetraethylammonium (TEA) trigger bursts of electrical activity and oscillations of intracellular free Ca2+ concentration ([Ca2+]i) in the presence of 100 microM tolbutamide. The [Ca2+]i was monitored from single islets of Langerhans using fura-2 microfluorescence techniques. Both the high-Ca(2+)- and Bay-K-8644-evoked [Ca2+]i oscillations overshot the [Ca2+]i recorded in tolbutamide. Nifedipine (10-20 microM) caused an immediate membrane hyperpolarization, which was followed by a slow depolarization to a level close to the burst active phase potential. The latter depolarization was accompanied by suppression of spiking activity. Exposure to high Ca2+ in the presence of nifedipine caused a steady depolarization of approximately 8 mV. Ionomycin (10 microM) caused membrane hyperpolarization in the presence of 7.7 mM Ca2+, which was not abolished by nifedipine. Charybdotoxin (CTX, 40-80 nM), TEA (2 mM) and quinine (200 microM) did not suppress the high-Ca(2+)-evoked bursts. It is concluded that: (1) the channel underlying the burst is sensitive to [Ca2+]i rises mediated by Ca2+ influx through L-type Ca2+ channels, (2) both the ATP-dependent K+ channel and the CTX- and TEA-sensitive Ca(2+)-dependent K+ channel are highly unlikely to provide the pacemaker current underlying the burst.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucose depolarizes the pancreatic beta-cell and induces membrane potential oscillations, but the nature of the underlying oscillatory conductance remains unknown. We have now investigated the effects of the Ca2+ ionophore ionomycin and high external Ca2+ concentration ([Ca2+]o) on glucose-induced electrical activity and whole islet intracellular free Ca2+ concentration ([Ca2+]i), under conditions where the K(ATP) channel was blocked (100 microM tolbutamide or 4 microM glibenclamide). Raising [Ca2+]o to 10.2 or 12.8 mM, but not to 5.1 or 7.7 mM, turned continuous electrical activity into bursting activity. High [Ca2+]o (12.8 mM) regenerated a pattern of fast [Ca2+]i oscillations overshooting the levels recorded in tolbutamide. Ionomycin (10 microM) raised the [Ca2+]i and synergized with 5.1 mM Ca2+ to hyperpolarize the beta-cell membrane. The data indicate that a [Ca2+]i-sensitive and sulphonylurea-insensitive oscillatory conductance underlies the beta-cell bursting activity.
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Pancreatic beta cells, tightly organized in the islet of Langerhans, secrete insulin in response to glucose in a calcium-dependent manner. The calcium input required for this secretory activity is thought to be provided by an oscillatory electrical activity occurring in the form of "bursts" of calcium action potentials. The previous observation that islet intracellular free Ca2+ levels undergo spontaneous oscillations in the presence of glucose, together with the fact that islet cells are coupled through gap junctions, hinted at a highly effective co-ordination between individual islet cells. Through the use of simultaneous recordings of intracellular calcium and membrane potential it is now reported that the islet calcium waves are synchronized with the beta cell bursting electrical activity. This observation suggests that each calcium wave is due to Ca2+ entering the cells during a depolarized phase of electrical activity. Moreover, fura-2 fluorescence image analysis indicates that calcium oscillations occur synchronously across the whole islet tissue. The maximal phase shift between oscillations occurring in different islet cells is estimated as 2 s. This highly co-ordinated oscillatory calcium signalling system may underlie pulsatile insulin secretion and the islet behaviour as a secretory "syncytium". Since increasing glucose concentration lengthens calcium wave and burst duration without significantly affecting wave amplitude, we further propose that it is the fractional time at an enhanced Ca2+ level, rather than its amplitude, that encodes for the primary response of insulin-secreting cells to fuel secretagogues.
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Intracellular Ca2+ levels were monitored in single, acutely isolated mouse islets of Langerhans by dual emission Indo-1 fluorometry. High-frequency (3.1 min-1) [Ca2+]i oscillations with a brief rising time (1-2 s) and 10 s half-width ('fast' oscillations) were detected in 11 mM glucose. Raising the glucose concentration to 16.7 mM increased the duration of these oscillations, which were otherwise absent in 5.5 mM glucose. [Ca2+]i waves of lower frequency (0.5 min-1) and longer rising time ('slow' oscillations) were also recorded. The data indicate that "fast" oscillations are directly related to beta-cell bursting electrical activity, and suggest the existence of extensive networks of electrically coupled cells in the islet.
Acetylcholine (1-10 microM) depolarized the membrane and stimulated glucose-induced bursts of electrical activity in mouse pancreatic B-cells. The acetylcholine effects were mimicked by muscarine while nicotine had no effect on membrane potential. Pirenzepine, an antagonist of the classical M1-type muscarinic receptors, but not gallamine (1-100 microM), an antagonist of the classical M2-type receptors, antagonized the acetylcholine action on glucose-induced electrical activity (IC50 = 0.25 microM). Bethanechol, an agonist of the classical M2-type muscarinic receptors, was approximately 100 times less effective than acetylcholine in stimulating the electrical activity. In addition, acetylcholine (1 microM) induced a marked increase (25%) in input resistance to the B-cell membrane. The results indicate that acetylcholine exerted its effects on the B-cell membrane by inhibiting K+ conductance via activation of a muscarinic receptor subtype distinct from the classical M2-type receptor.
The effects of forskolin on electrical coupling among pancreatic beta-cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet beta-cells. Intracellular injection of a current pulse (delta I) elicited a membrane response delta V1 in the injected cell and also a response delta V2 in a nearby beta-cell confirming the existence of cell-to-cell electrical coupling among islet beta-cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the beta-cell membrane K+ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in beta-cell input resistance (delta V2/delta I) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio delta V2/delta V1 provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet beta-cells.
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