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R Zorec

Publications and source records attributed to R Zorec.

At least 37 records · Page 2Linked to original sources
PubMed1995

Brefeldin A and a synthetic peptide to ADP-ribosylation factor (ARF) inhibit regulated exocytosis in melanotrophs.

We investigated the role of ADP-ribosylation factor (ARF) in regulated exocytosis in patch-clamped rat melanotrophs. Addition of brefeldin A (BFA) to inhibit activation of endogenous ARF protein was found to attenuate regulated secretory activity monitored as changes in membrane capacitance (Cm). A synthetic peptide to amino acids 46-61 of ARF (P-14) was also found to inhibit Ca(2+)-induced secretory activity in these cells. This inhibition was not apparent with a scrambled amino acid sequence of ARF-P14 peptide. This paper provides the first patch-clamp study to suggest that the small GTP-binding protein ARF is required to trigger release of secretory granules from rat pituitary melanotrophs.

ADP-Ribosylation Factors↗
PubMed1995

Intracellular Cl- modulates Ca2+-induced exocytosis from rat melanotrophs through GTP-binding proteins.

We used the whole-cell patch-clamp technique to monitor changes in membrane capacitance (Cm) to study the influence of cytosolic concentration ([Cl-]i) on the secretory activity of rat melanotrophs. The sensitivity of the secretory machinery to Ca2+ was enhanced in the presence of a high [Cl-]i. The free concentration of Ca2+ required for half-maximal secretory activity was reduced from 3.2microM at 4mM [Cl-]i to 0.7microM at 154mM [Cl-]i. To study whether the modulation of secretory activity by Cl- involves guanosine 5'-triphosphate-(GTP-) binding proteins, cells were dialysed with non-hydrolysable GTP and GDP analogues, fluoroaluminate (AlF4(-)), or were pretreated with pertussis toxin. With guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S], 100microM) the maximal rate of Cm increase (dCm/dt) was enhanced at 4 and 14mM [Cl-]i, but it was not affected at 154mM [Cl-]i. In contrast, the secretory response, measured as a percentage of resting Cm 10min after the start of recordings, was reduced at 154mM [Cl-]i, but not affected at 4mM [Cl-]i. Only with 154mM [Cl-]i did intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) (GDP[beta-S], 500microM) inhibit dCm/dt as well as relative secretory responses. The presence of AlF4(-) (30microM) or a 7-h pretreatment of cells with pertussis toxin (250ng/ml) significantly reduced both the maximal dCm/dt and relative secretory responses, but only in the presence of 154mM [Cl-]i. Since the effects of GDP[beta-S], AlF4(-), and pertussis toxin pretreatment were only detected with a high [Cl-]i, we conclude that modulation by Cl- of secretory activity of rat melanotrophs is mediated through GTP-binding proteins. Furthermore, the effects of AlF4(-) and pertussis toxin indicate a role of heterotrimeric GTP-binding proteins in the secretory activity of melanotrophs.

Aluminum Compounds↗
PubMed1995

Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.

Animals↗
PubMed1994

The separation of exocytosis from endocytosis in rat melanotroph membrane capacitance records.

1. Using the patch-clamp technique, we have monitored the secretory activity of single rat melanotrophs. Changes in membrane capacitance (Cm) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measure the amplitude of discrete steps in Cm. Records of Cm were converted into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in Cm was easily distinguished from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer-based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dCm/dt versus Cm, from which the magnitude and direction of transients were determined. Transients due to 2 fF steps (equivalent to a signal-to-noise ratio of 1) were detected with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detections of 0.075 S-1 was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs with an anti-adrenocorticotrophin (ACTH) antibody. Secretory organelles in cultured and non-cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organelles of similar diameter stained positively with the anti-ACTH antibodies. 4. Small discrete steps in Cm, recorded with the whole-cell configuration and loosely buffered cytosolic calcium, were similar to the estimated Cm of secretory organelles from morphological data. Thus, measured discrete steps in Cm reflect interactions between single organelle size and plasma membrane. Exocytotic and endocytotic steps were found to be of similar size. 5. To separate exocytosis from endocytosis in Cm records, we assumed that the rates of exocytosis and endocytosis were related to the respective frequencies of discrete steps in Cm. A relationship between the frequency of exocytotic, but not endocytotic events, and the rate of change in Cm was observed. Thus, under our experimental conditions, an increase in Cm could be explained by an increased rate of exocytosis in rat melanotrophs.

Animals↗
PubMed1994

Rab3 proteins: key players in the control of exocytosis.

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Animals↗
PubMed1994

Magnetic detection of a single action potential in Chara corallina internodal cells.

The electrical activity that occurs in plants has not yet been detected magnetically. Magnetic detection of electrical activity in some animal as well as in human cells and organs, on the other hand, is an established research method. Our experiments demonstrate the propagation of a single action potential in the internodal cell of the green algae Chara corallina, measured magnetically. The propagation velocity and the intracellular current were determined.

Action Potentials↗
PubMed1993

Rapid pressure driven exocytosis-endocytosis cycle in a single plant cell. Capacitance measurements in aleurone protoplasts.

The whole-cell patch-clamp technique has been used to increase intracellular pressure via the pipette while monitoring changes in membrane capacitance (related to membrane area) in single aleurone protoplasts. Increased pressure increased membrane capacitance and diameter, upon the release of pressure both parameters returned to resting levels. Pressure also reversibly increased membrane conductance. Comparison between diameter and capacitance shows that the capacitance changes are due to changes in cell surface area. These results show that pressure can rapidly alter the balance between exo- and endocytosis. Pressure-induced changes in conductance are suggested to be due to insertion/withdrawal of channels with the exo-/endocytosed membrane.

Electric Conductivity↗
PubMed1993

Inhibition of Rab3B expression attenuates Ca(2+)-dependent exocytosis in rat anterior pituitary cells.

Low-molecular-weight GTP-binding proteins (small G proteins) of the Rab family have been proposed to act as central regulators of vesicular traffic, and proteins of the Rab3 subfamily (Rab3A, B, C and D) are thought to be associated with membrane vesicles or granules undergoing exocytotic fusion with the plasma membrane. Rab3A is highly expressed in brain, whereas Rab3B is the major form found in rat anterior pituitary gland. We report here that antisense oligonucleotides against Rab3B, introduced into pituitary cells using the whole-cell patch clamp technique, specifically and reversibly block expression of Rab3B. We find that calcium-dependent exocytosis is inhibited without affecting endocytosis. Antisense oligonucleotides directed against Rab3A have no effect. Our results indicate that Rab3B is likely to be a key intracellular signalling molecule which can control exocytosis downstream of other calcium-dependent processes in anterior pituitary cells.

Animals↗
PubMed1993

Properties of acetylcholine receptors in adult rat skeletal muscle fibers in culture.

The distribution and biophysical properties of acetylcholine receptors were studied, using morphological and patch-clamp techniques, in adult rat skeletal muscle fibers dissociated by collagenase and maintained in culture. Up to ten hours after dissociation, there were no changes in either the distribution or the biophysical properties of junctional acetylcholine receptors. In long-term culture (5 to 14 days), a new type of acetylcholine receptor was inserted all over the muscle fibers; the channel properties were characterized by a longer open time and a smaller conductance, similar to what has been observed in in vivo denervated muscles. Using autoradiography, we found that during culture an impaired incorporation of new acetylcholine receptors in the former endplates caused a progressive decrease in the density of junctional acetylcholine receptors. This contrasts with muscle fibers denervated in vivo, where the density of receptors does not change after denervation.

Animals↗
PubMed1993

A new approach to separation of voltage-activated Ca currents in rat melanotrophs.

The whole cell patch clamp technique was used to study voltage-activated calcium currents in rat melanotrophs. Voltage ramps of the same amplitude and of different durations were applied to separate Ca currents with different electrophysiological properties. The application of those with voltage gradient of 0.5 V/s resulted in a clear separation of two inward current components, activated at -55 +/- 5 mV and -24 +/- 5 mV respectively, corresponding to low (LVA) and high (HVA) voltage-activated currents. The proportion of cells displaying such current separation was reduced either at voltage gradients of 0.053 V/s or 1.6 V/s. The application of nitrendipine and nicardipine resulted in a blockage of both currents, where the HVA current component was more sensitive to the agents. The sensitivity of both Ca currents to dihydropyridine antagonists suggests that these currents may be mediated by subtypes of a dihydropyridine sensitive Ca channel.

Animals↗
PubMed1993

Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy.

The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2',7'-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH4Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH4Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na+, suggesting the presence of Na+/H+ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na+ from the bathing medium results in a decrease in resting [Ca2+]i, paralleled by a reduction in pHi. This suggests a role for Na+/H+ exchange in determining resting [Ca2+]i.

Ammonium Chloride↗
PubMed1992

Cytosolic chloride ions stimulate Ca(2+)-induced exocytosis in melanotrophs.

We used the whole-cell patch-clamp technique to study the secretory activity of single cells by monitoring changes in membrane capacitance [Neher, E. and Marty, A. (1982) Proc. Natl. Acad. Sci. USA 79, 523-535] in anterior pituitary cells. Unexpectedly we have observed that increasing intracellular chloride ions stimulate Ca(2+)-induced exocytosis in a dose-dependent fashion (Kd = 12 mM). These results demonstrate a role of cytosolic chloride ions in the regulation of exocytotic secretion in anterior pituitary cells. It is suggest that chloride channels, in addition to playing a part in regulating membrane electrical activity [Korn, S.J., Bolden, A. and Horn, A. (1991) J. Physiol. 439, 423-437; Penner, R., Matthews, G. and Horn, A. (1988) Nature 334, 499-504] and cytosolic pH [Kaila, K. and Voipio, J. (1987) Nature 330, 163-165], are also involved in the modulation of cytosolic chloride concentration and thus in the control of exocytosis.

Animals↗
PubMed1992

Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels.

The use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of ACh-activated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 microM) or MSF (5 mM) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence less than 10%. In the presence of 1.5 mM MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 microM PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mM MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.

Acetylcholine↗
PubMed1991

Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance.

The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.

Animals↗