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Papers by Eduardo Saldaña Rojas
Endocrinology, 1997
In cultured rat pituitary gonadotrophs, GnRH-induced oscillations in cytosolic calcium concentrat... more In cultured rat pituitary gonadotrophs, GnRH-induced oscillations in cytosolic calcium concentration ([Ca 2ϩ ] i ) are associated with periodic membrane hyperpolarization. The hyperpolarizing waves are secondary to the activation of apamin-sensitive Ca 2ϩ -activated K ϩ channels that account for a single class of 125 I-apamin binding sites present in these cells. In a substantial fraction of gonadotrophs, however, we observed a Ca 2ϩ -controlled oscillatory current that was resistant to apamin, even at concentrations five orders of magnitude higher than the dissociation constant (K d ) observed in the binding experiments. With the K ϩ in the pipette, the apamin-resistant current showed a reversal potential of Ϫ42 mV, nearly 40 mV more positive than that of the apamin-sensitive current. With Cs ϩ in place of K ϩ in the pipette solution, both the size of the apamin-insensitive current and its reversal potential remained unchanged. Ion substi-
Biological Research, 2006
We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreati... more We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.
Nature, 1967
ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium mo... more ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium movements1, there has been little explicit discussion of how these ion movements may be initiated, or of the chemical processes which may permit them to occur2. Because it is not possible to identify the molecules involved by measuring potentials and associated ion movements, there is a
Land Use Policy, 1984
The main effects of existing socioeconomic arrangements for agricultural land development in the ... more The main effects of existing socioeconomic arrangements for agricultural land development in the Eastern Caribbean are discussed and changes aimed at improving both the efficiency and squity of the land development process are identified. The author argues that the existing 'plantations-small holders' structure is incapable of providing an efficient way of developing the available land resources and of improving the standard of living for the vast rural population. A new set of export oriented agrlcultural enterprises is pinpointed as a requirement in any viable solution aimed at taking land away from its present role as a resource for survival for the many and an economic benefit for the few and to reinstate agricultural lands as one of the key resources for more egalitarian, environmentally sound and efficient sociosconomic devel-
Habitat International, 1984
Neuroendocrinology, 1996
The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 li... more The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of approximately 200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of -10 to +60 mV from a holding potential of -90 mV elicited outward current with early transient and sustained components. The transient current peaked 2-10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+ -activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 mu M), and (4) prolonged by the endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin (1 mu M). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30-60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to gamma-aminobutyric acid (GABA, 100 mu M), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells remains elusive.
FEBS Letters, 1987
The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffi... more The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffin granules and fusion of granule ghosts involves specific interactions with the lipid component of the membrane. To study the details of these interactions we measured synexin-induced changes in capacitance of phosphatidylserine bilayers formed at the tip of a patch pipet using the double-dip method. Provided calcium was present in the solution filling the pipet (10-50 mM) stable phosphatidylserine bilayers were easily formed. Addition of synexin (0.1 pg/ml) to an external medium lacking added calcium induced no measurable changes in either bilayer resistance (10-30 GQ) or displacement current across the membrane. However, addition of calcium (0.1-2.5 mM) in the presence of synexin in the external solution caused a marked increase in the size and time constant of decay of the displacement current. From the steady-state value of the current we calculated a 5-fold decrease in resistance and from the charge displaced during the voltage-clamp pulses we calculated a IO-fold increase in membrane capacitance (from 20 to 200 fF). The size of the synexin-specific charge displacement in one direction during a pulse was always equal to the charge returning to the original configuration after the pulse. The synexin-specific transfer of charge reached saturation when the pipet potential was taken to a sufficient positive or negative value. These properties of the extra charge movement support our view that in the presence of calcium the cytosolic protein synexin penetrates into the bilayer. It is possible that these properties may be related to the mechanism by which synexin promotes membrane fusion in natural membranes.
FEBS Letters, 1986
Measurements of the effects of NH,/NHt on glucose-induced electrical activity in /J-cells from mi... more Measurements of the effects of NH,/NHt on glucose-induced electrical activity in /J-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH,/NHt (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the /I-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH,Cl initially stumulated electical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH&l there was a drop in pH, followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca2+]-dependent K+ channel in the /?-cell membrane is activated by a rise in cytosolic pH.
FEBS Letters, 1989
Acetylcholine (1-10 microM) depolarized the membrane and stimulated glucose-induced bursts of ele... more 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.
Cellular and Molecular Neurobiology, 1995
1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the ol... more 1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the A/3P peptide in amyloid plaques.
Endocrinology, 1997
In cultured rat pituitary gonadotrophs, GnRH-induced oscillations in cytosolic calcium concentrat... more In cultured rat pituitary gonadotrophs, GnRH-induced oscillations in cytosolic calcium concentration ([Ca 2ϩ ] i ) are associated with periodic membrane hyperpolarization. The hyperpolarizing waves are secondary to the activation of apamin-sensitive Ca 2ϩ -activated K ϩ channels that account for a single class of 125 I-apamin binding sites present in these cells. In a substantial fraction of gonadotrophs, however, we observed a Ca 2ϩ -controlled oscillatory current that was resistant to apamin, even at concentrations five orders of magnitude higher than the dissociation constant (K d ) observed in the binding experiments. With the K ϩ in the pipette, the apamin-resistant current showed a reversal potential of Ϫ42 mV, nearly 40 mV more positive than that of the apamin-sensitive current. With Cs ϩ in place of K ϩ in the pipette solution, both the size of the apamin-insensitive current and its reversal potential remained unchanged. Ion substi-
Biological Research, 2006
We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreati... more We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.
Nature, 1967
ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium mo... more ALTHOUGH there is evidence that axonal membrane potentials are related to sodium and potassium movements1, there has been little explicit discussion of how these ion movements may be initiated, or of the chemical processes which may permit them to occur2. Because it is not possible to identify the molecules involved by measuring potentials and associated ion movements, there is a
Land Use Policy, 1984
The main effects of existing socioeconomic arrangements for agricultural land development in the ... more The main effects of existing socioeconomic arrangements for agricultural land development in the Eastern Caribbean are discussed and changes aimed at improving both the efficiency and squity of the land development process are identified. The author argues that the existing 'plantations-small holders' structure is incapable of providing an efficient way of developing the available land resources and of improving the standard of living for the vast rural population. A new set of export oriented agrlcultural enterprises is pinpointed as a requirement in any viable solution aimed at taking land away from its present role as a resource for survival for the many and an economic benefit for the few and to reinstate agricultural lands as one of the key resources for more egalitarian, environmentally sound and efficient sociosconomic devel-
Habitat International, 1984
Neuroendocrinology, 1996
The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 li... more The expression and function of large-conductance Ca2+ -activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of approximately 200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of -10 to +60 mV from a holding potential of -90 mV elicited outward current with early transient and sustained components. The transient current peaked 2-10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+ -activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 mu M), and (4) prolonged by the endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin (1 mu M). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30-60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to gamma-aminobutyric acid (GABA, 100 mu M), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells remains elusive.
FEBS Letters, 1987
The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffi... more The mechanism by which synexin mediates calcium-dependent aggregation of medullary cell chromaffin granules and fusion of granule ghosts involves specific interactions with the lipid component of the membrane. To study the details of these interactions we measured synexin-induced changes in capacitance of phosphatidylserine bilayers formed at the tip of a patch pipet using the double-dip method. Provided calcium was present in the solution filling the pipet (10-50 mM) stable phosphatidylserine bilayers were easily formed. Addition of synexin (0.1 pg/ml) to an external medium lacking added calcium induced no measurable changes in either bilayer resistance (10-30 GQ) or displacement current across the membrane. However, addition of calcium (0.1-2.5 mM) in the presence of synexin in the external solution caused a marked increase in the size and time constant of decay of the displacement current. From the steady-state value of the current we calculated a 5-fold decrease in resistance and from the charge displaced during the voltage-clamp pulses we calculated a IO-fold increase in membrane capacitance (from 20 to 200 fF). The size of the synexin-specific charge displacement in one direction during a pulse was always equal to the charge returning to the original configuration after the pulse. The synexin-specific transfer of charge reached saturation when the pipet potential was taken to a sufficient positive or negative value. These properties of the extra charge movement support our view that in the presence of calcium the cytosolic protein synexin penetrates into the bilayer. It is possible that these properties may be related to the mechanism by which synexin promotes membrane fusion in natural membranes.
FEBS Letters, 1986
Measurements of the effects of NH,/NHt on glucose-induced electrical activity in /J-cells from mi... more Measurements of the effects of NH,/NHt on glucose-induced electrical activity in /J-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH,/NHt (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the /I-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH,Cl initially stumulated electical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH&l there was a drop in pH, followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca2+]-dependent K+ channel in the /?-cell membrane is activated by a rise in cytosolic pH.
FEBS Letters, 1989
Acetylcholine (1-10 microM) depolarized the membrane and stimulated glucose-induced bursts of ele... more 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.
Cellular and Molecular Neurobiology, 1995
1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the ol... more 1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the A/3P peptide in amyloid plaques.