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Biomedical subjects

E Rojas

Publications and source records attributed to E Rojas.

At least 145 records · Page 8Linked to original sources
PubMed1991

[Metacyclics of Leishmania mexicana in the hypopharynx of Lutzomyia youngi experimentally infected].

Although it has been postulated that the natural transmission of leishmaniasis occurs by regurgitation of the parasites from contaminated proboscis of phlebotomine vector recent experimental results seems to oppose this thesis. Wild-caught and laboratory-reared females of Lutzomyia youngi, vector of Leishmania mexicana in the Venezuelan Andes, were infected on tarsal lesion of hamsters, inoculated 6 weeks previously s.c. with 2 x 10(4) amastigotes of L. mexicana s.l., isolated from Venezuelan case of diffuse leishmaniasis. The insects were kept at 23 degrees C and 80% R.H., on a 50% (v:v) solution of "unrefined" cane sugar. The parasites developed through 3 stages: i) differentiation of the amastigotes and exponential growth in the number of promastigotes, ending between 60 and 108 hrs, with a massive fecal elimination of large promastigotes; ii) a stationary phase in growth with flagellar adhesion to the cuticle of the stomodeum and synthesis of a gel that formed a plug between 60 and 120 hrs; iii) differentiation of metacyclics, which invade the hypopharyngeal duct in 7% of the insects, from the 5th day post-infection, and most frequently between the 6th and 9th day. The metacyclics measured 4.2/microns in body length, 1.07/microns in maximum with and the flagellum was 8.8/microns long. The parasites swam freely in the saliva of the hypopharyngeal duct (lumen 6/microns in diameter), from the apex to union with the salivary duct, without invading the latter. Similar results has been obtained in the same vector experimentally infected with other two strains of L. mexicana isolated respectively from wild Lu. flaviscutellata and from another case of diffuse leishmaniasis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Calcium-activated endonexin II forms calcium channels across acidic phospholipid bilayer membranes.

Human endonexin II (annexin V) and recombinant human endonexin II can be activated by Ca2+ to interact with acidic phospholipid bilayers formed at the tip of a patch pipette. Once associated with the bilayer, endonexin II forms voltage-gated channels which are selective for divalent cations according to the following series Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+. However, endonexin II also expresses a selective affinity for Ca2+ which is manifest by an observed reduced current through the open channel when Ca2+ is the charge carrier. La3+ blocks endonexin II channels, as it does synexin (annexin VII) and other types of Ca2+ channels. However, as with synexin, the dihydropyridine Ca2+ channel antagonist nifedipine does not affect endonexin II channel activity. Endonexin II channels are also permeant to Li+, Cs+, Na+, and to a lesser extent, K+, resembling in this manner Ca2+ release channels from sarcoplasmic reticulum. Indeed, the low affinity of endonexin II channels for such ions as Cs+ or Li+ have allowed us to use these cations for measurement of the kinetic properties of the channel, with minimal concerns for the ion/channel interactions observed with the physiological substrate, Ca+. Finally, we observed that endonexin II channel activity always occurred in bursts, making necessary the use of two exponential functions to fit open- and closed-time histograms. We conclude from these data that the domain responsible for endonexin II channel activity, first observed by ourselves in the homologue synexin, is probably the C-terminal tetrad repeat common to both molecules.

Amino Acid Sequence↗
PubMed1990

Kinetic characteristics of calcium-dependent, cholinergic receptor controlled ATP secretion from adrenal medullary chromaffin cells.

Adrenal chromaffin cells secrete catecholamines (CA) and ATP in response to acetylcholine (ACh) and high [K+]o. The release process is relatively fast making it difficult to measure the early phase of the secretory response. Recently we were able to resolve the time course of the secretory response by measuring the release of ATP using luciferin-luciferase included in the extracellular medium. For the three secretagogues studied, ACh, nicotine and high [K+]o, the early phase of release followed a complex kinetics. Allowing for an initial delay of the secretory response, the kinetics could be described as the sum of two power exponential processes. Increasing the temperature from 23 to 37 degrees C induced a marked decrease in the two time constants needed to fit the early time course of the ATP secretion. The activation energies, estimated from Arrhenius plots, were approx. 20 and 16 kcal/mol for both phases of ATP release induced by either cholinergic agonists or high [K+]o. These results suggest that cholinergic receptor activation and membrane depolarization induce ATP (and CA) secretion through a common pathway. The initial delay in the onset of the secretory response decreased with increasing doses of secretagogue and with temperature. We propose that the delay preceding the actual onset of ATP release represents the time required for generation of intracellular second messengers. The effective concentration attained by these messengers depend apparently on both receptor occupancy by the agonist and the ensuing Ca2+ channel activation.

Acetylcholine↗
PubMed1990

A new class of calcium channels activated by glucose in human pancreatic beta-cells.

Single calcium-channel currents were recorded from membrane patches of cultured beta-cells dissociated from human islets of Langerhans. In the absence of exogenous glucose, low frequency spontaneous calcium-channel openings of small amplitude (-0.34 +/- 0.02 pA at 0 mV pipet potential) were observed in all membrane patches examined (25 mM Ca2+ in the patch pipet). The frequency of channel openings was rather insensitive to the membrane potential across the patch (range from ca 0 to 60 mV pipet potential; chord conductance 4.9 +/- 0.2 pS). Addition of glucose induced a dose-dependent increase in the frequency of openings of the Ca2(+)-channel (from now on referred to as the CaG-channel). A few minutes after the addition of glucose (greater than or equal to 11 mM), bursts of action potentials were often observed which were elicited only if Ca2+ was present in the solution bathing the beta-cells. Application of glucose in the presence of mannoheptulose (11 mM), a blocker of the hexokinase controlling the first stage of glycolysis, had no effect and the activity of the CaG-channel remained at its resting level. The readily permeant mitochondrial substrate 2-keto-isocaproate (KIC, 10 mM) was as effective as glucose in eliciting action potentials from cells forming part of cell aggregates. The activity of the CaG-channel was significantly increased by KIC (11 mM). Although spike and Ca2(+)-channel activity were markedly stimulated by glucose or KIC in all cells examined, regular bursts of action potentials were seen only if the patch was formed on beta-cells which were part of a cell aggregate. Mannoheptulose (11 mM) prevented the activation of the CaG-channel by glucose (11 mM) but not by KIC (11 mM). Once activated, the CaG-channel remained active even after excision of the patch. We propose that the physiological control of this Ca2(+)-channel is mediated by one or more products of glucose metabolism.

Action Potentials↗
PubMed1990

Quinine blocks the high conductance, calcium-activated potassium channel in rat pancreatic beta-cells.

The [Ca2+]i-activated K+-channel, one of the 3 K+ -channels described in pancreatic beta-cells, is a high conductance, voltage-dependent K+-channel. Quinine, known to block [Ca2+]i-activated K(+)-channels in other cells, has been described to block the silent phase between the bursts of glucose-evoked electrical activity in mouse pancreatic beta-cells, and to inhibit K+ efflux from rat pancreatic islets. We report here that quinine blocks the [Ca2+]i-activated K(+)-channel in rat pancreatic beta-cells from the external side of the membrane. We also show that the blockade is characterized by fast flickering of the K(+)-channel between the open and closed state. Mean open and closed times within bursts were found to be exponentially distributed, suggesting that the blockade by quinine involves obstruction on the K(+) flow through the open to be exponentially distributed, suggesting that the blockade by quinine involves obstruction on the K+ flow through the open channel.

Animals↗
PubMed1990

Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs.

The relationships between the activation status of voltage-sensitive Ca2+ channels and secretory responses were analyzed in perfused rat gonadotrophs during stimulation by high extracellular K+ concentration ([K+]e) or the physiological agonist, gonadotropin-releasing hormone (GnRH). Increase of [K+]e to 50 mM evokes an on-off secretory response, with a rapid rise in luteinizing hormone (LH) secretion to a peak at 35 sec (on response) followed by an exponential decrease to the steady-state level. Cessation of K+ stimulation elicits a transient (off) response followed by an exponential decrease to the basal level. The LH response to high [K+]e is nifedipine-sensitive and its amplitude depends on membrane potential. There is a close relationship between the LH secretory response to high [K+]e and the amplitude of the inward Ca2+ current measured at 100 msec in whole-cell patch clamp experiments. In addition, the profile of the LH secretory response is similar to that of the response of intracellular Ca2+ concentration ([Ca2+]i) in K(+)-stimulated cells. In Ca2(+)-deficient medium, the effect of high [K+]e is abolished; subsequent elevation of [Ca2+]e during the K+ pulse is followed by restoration of the on response, but with reduced magnitude. Agonist stimulation during the steady-state phase of the [K+]e pulse or after repetitive stimulation by high [K+]e elicited biphasic [Ca2+]i and secretory responses with a significantly reduced plateau phase; conversely, K(+)-induced LH release was reduced in cells treated with desensitizing doses of GnRH. These findings indicate that depolarization-induced changes in the status of voltage-sensitive Ca2+ channels determine the profiles of [Ca2+]i and LH responses to stimulation by high [K+]e; the initial activation of dihydropyridine-sensitive Ca2+ channels is clearly dependent on membrane potential, whereas their subsequent inactivation depends on increased [Ca2+]i. Such inactivation of voltage-sensitive Ca2+ channels also occurs during GnRH action and may represent an additional regulatory mechanism to limit the entry of extracellular Ca2+ during prolonged or frequent agonist stimulation.

Animals↗
PubMed1990

Generation and amplification of the cytosolic calcium signal during secretory responses to gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) stimulates characteristic biphasic increases in cytosolic calcium concentration ([Ca2+]i) and in luteinizing hormone (LH) release in cultured gonadotrophs, with an early peak followed by a prolonged plateau in both responses. Analysis of [Ca2+]i by dual-wavelength fluorimetric assay and of LH release at 5-sec intervals in perifused pituitary cells revealed increases in both responses within a few seconds of exposure to GnRH. The maximum elevation of [Ca2+]i occurred within 20 sec, and the peak gonadotropin release in 35 sec; the total duration of the spike phase for both [Ca2+]i and LH release was 2.5 min. Under extracellular Ca2(+)-deficient conditions, the GnRH-induced peak in [Ca2+]i was reduced by about 20% and the plateau phase was abolished. Concomitantly, the magnitude of the acute phase of LH release was reduced by 40% and that of the second phase by about 90%. Recovery of the plateau phase of LH release occurred within 25 sec after addition of 1.25 mM Ca2+ to Ca2(+)-deficient medium. In a dose-dependent manner, the non-selective Ca2+ channel blockers Co2+ and Cd2+ reduced the Ca2+ current measured by whole-cell recording in pituitary gonadotrophs and abolished the extracellular Ca2(+)-dependent component of LH release. The selective calcium channel blocker, nifedipine, decreased the magnitude of the Ca2+ current and reduced the plateau phase of LH release by 50%; conversely, the dihydropyridine agonist methyl, 1,4,dihydro-2,6-dimethyl 3-nitro-4-(2-trifluorome) (Bay K 8644) consistently enhanced the amplitudes of both Ca2+ current and GnRH-induced LH release. These data reveal a close temporal correlation between changes in [Ca2+]i and LH release during GnRH action, with Ca2+ mobilization during the spike phase and Ca2+ influx through dihydropyridine-sensitive and insensitive sets of receptor-operated calcium channels during the spike and plateau phases. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+ is consistent with amplification of the [Ca2+]i signal in agonist-stimulated gonadotrops.

Animals↗
PubMed1989

Desensitization of pituitary gonadotropin secretion by agonist-induced inactivation of voltage-sensitive calcium channels.

Gonadotropin-releasing hormone (GnRH) stimulates calcium mobilization and influx in pituitary gonadotrophs, and agonist-induced calcium entry through voltage-sensitive channels (VSCC) is required for the maintenance of gonadotropin secretion. However, prolonged or frequent exposure to GnRH attenuates the extracellular Ca2+-dependent cytosolic Ca2+ signal and diminishes hormone secretion. Measurements of membrane Ca2+ currents revealed significant impairment of VSCC activity in gonadotrophs during desensitization by GnRH. VSSC were also inactivated in a calcium-dependent manner during exposure to high K+. Prolonged inactivation of such Ca2+ channels by high K+ reduced the calcium and secretory responses to GnRH and vice versa. The calcium-dependent inactivation of VSCC during GnRH action appears to be a primary factor in the onset of desensitization in pituitary gonadotrophs. This mechanism could also account for the development of agonist-induced refractoriness in other calcium-regulated target cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Muscarinic receptor modulation of glucose-induced electrical activity in mouse pancreatic B-cells.

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.

Acetylcholine↗
PubMed1989

Effects of calcium and Bay K-8644 on calcium currents in adrenal medullary chromaffin cells.

The kinetic and steady-state characteristics of calcium currents in cultured bovine adrenal chromaffin cells were analyzed by the patch-clamp technique. Whole cell inward Ca2+ currents, recorded in the presence of either 5.2 or 2.6 mM Ca2+ exhibited a single, noninactivating component. To analyze the effects of Ca2+ and Bay K-8644 on the kinetics of the Ca2+ currents, we used a modified version of the Hodgkin-Huxley empirical model. At physiological [Ca2+] (2.5 mM) the midpoint of the steady-state Ca2(+)-channel activation curve lay at -6.9 mV. Increasing the [Ca2+] to 5.2 mM shifted the midpoint by -4.3 mV along the voltage axis. At the midpoint, changes in potential of 7.8 mV (for 5.2 mM Ca2+) and 9.2 mV (for 2.5 mM Ca2+) induced an e-fold change in the activation of the current. Increasing [Ca2+]o from 2.5 to 5.2 mM induced a marked increase in the rate constant for turning on the Ca2+ permeability. Conductances were estimated from the slope of the linear part of the current-voltage relationships as 8.7 and 4.2 nS in the presence of 5.2 and 2.5 mM Ca2+, respectively. Incubation of the cells in the presence of Bay K-8644 at increasing concentrations from 0.001 to 0.1 microM increased the slope conductance from 4.2 to 9.6 nS. Further increases in the concentration of Bay K-8644 from 1 to 100 microM induced a marked reduction in the conductance to 1.1 nS. In the presence of Bay K-8644 (0.1 microM) the midpoint of the activation curve was shifted by 6.1 mV towards more negative potentials, i.e., from -6.9 to -13 mV. At the midpoint potential of -13 mV, a change in potential of 6.9 mV caused an e-fold change in Ca2+ permeability. The kinetic analysis showed that Bay K-8644 significantly reduced the size of the rate constant for turning off the Ca2+ permeability.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Calcium channel activity of purified human synexin and structure of the human synexin gene.

Synexin is a calcium-dependent membrane binding protein that not only fuses membranes but also acts as a voltage-dependent calcium channel. We have isolated and sequenced a set of overlapping cDNA clones for human synexin. The derived amino acid sequence of synexin reveals strong homology in the C-terminal domain with a previously identified class of calcium-dependent membrane binding proteins. These include endonexin II, lipocortin I, calpactin I heavy chain (p36), protein II, and calelectrin 67K. The Mr 51,000 synexin molecule can be divided into a unique, highly hydrophobic N-terminal domain of 167 amino acids and a conserved C-terminal region of 299 amino acids. The latter domain is composed of alternating hydrophobic and hydrophilic segments. Analysis of the entire structure reveals possible insights into such diverse properties as voltage-sensitive calcium channel activity, ion selectivity, affinity for phospholipids, and membrane fusion.

Amino Acid Sequence↗