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R Zorec

Publications and source records attributed to R Zorec.

At least 19 recordsLinked to original sources
PubMed2005

FM1-43 measurements of local exocytotic events in rat melanotrophs.

We have explored the existence of fusion- and secretion-competent sites on the plasma membrane of peptide secreting rat pituitary melanotrophs at rest, and following stimulation with glutamate. We monitored changes in fluorescence of FM1-43, a styryl dye which labels plasma membrane. The results show spontaneous local increases in FM1-43 reporting changes in membrane surface area due to cumulative exocytosis. Addition of glutamate, further increased the occurrence of these events. Statistical analysis of local FM1-43 fluorescence changes suggests that this is due to the recruitment of inactive exocytotic domains and due to the stimulation of already active exocytotic domains.

Animals↗
PubMed2004

Automated high through-put colocalization analysis of multichannel confocal images.

The laser scanning confocal microscope (LSCM) generates images of multiple labelled fluorescent samples. Colocalization of fluorescent labels is frequently examined. Here we present an example where localization of fluorescent analogues of cloned protein were referenced to fluorescent antibodies directed against the proteins of cellular compartments. Colocalization is usually evaluated by visual inspection of signal overlap or by using commercially available software tools, but there are limited possibilities to automate the analysis of large amounts of data. We developed a simple tool using Matlab to automate the colocalization procedure and to exclude the biased estimations resulting from visual inspections of images. The script in Matlab language code automatically imports confocal images and converts them into arrays. The contrast of all images is uniformly set by linearly reassigning the values of pixel intensities to use the full 8-bit range (0-255). Images are binarized on several threshold levels. The area above a certain threshold level is summed for each channel of the image and for colocalized regions. As a result, count of pixels above several threshold levels in any number of images is saved in an ASCII file. In addition Pearson's r correlation coefficient is calculated for fluorescence intensities of both confocal channels. Using this approach quick quantitative analysis of colocalization of hundreds of images is possible. In addition, such automated procedure is not biased by the examiner's subject visualization.

Automation↗
PubMed2003

Apoptosis triggered redistribution of caspase-9 from cytoplasm to mitochondria.

Caspase-9 is an apoptosis initiator protease activated as a response to the mitochondrial damage in the cytoplasmic complex apoptosome. By fluorescence labelling of proteins, confocal microscopy and subcellular fractionations we demonstrate that caspase-9 is in the cytoplasm of non-apoptotic pituitary cells. The activation of apoptosis with rotenone triggers the redistribution of caspase-9 to mitochondria. Experiments using the general caspase inhibitor z-VAD.fmk and the specific caspase-9 inhibitor z-LEHD.fmk show that the caspase-9 redistribution is a regulated process and requires the activity of a caspase other than the caspase-9. We propose that this spatial regulation is required to control the activity of caspase-9.

Amino Acid Chloromethyl Ketones↗
PubMed2003

Properties of exocytotic response in vertebrate photoreceptors.

Synaptic transmission at the photoreceptor synapse is characterized by continuous release of glutamate in darkness. Release is regulated by the intracellular calcium concentration ([Ca2+]i). We here examined the physiological properties of exocytosis in tiger salamander (Ambystoma tigrinum) retinal rods and cones. Patch-clamp capacitance measurements were used to monitor exocytosis elicited by a rapid and uniform increase in [Ca2+]i by photolysis of the caged Ca2+ compound NP-EGTA. The amplitude of flash-induced increases in membrane capacitance (Cm) varied monotonically with [Ca2+]i beyond approximately 15 microM. The following two types of kinetic responses in Cm were recorded in both rods and cones: 1) a single exponential rise (39% of cells) or 2) a double-exponential rise (61%). Average rate constants of rapid and slow exocytotic responses were 420 +/- 168 and 7.85 +/- 5.02 s-1, respectively. The rate constant for the single exponential exocytotic response was 17.5 +/- 12.4 s-1, not significantly different from that of the slow exocytotic response. Beyond the threshold [Ca2+]i of approximately 15 microM, the average amplitude of rapid, slow, and single Cm response were 0.84 +/- 0.35, 0.82 +/- 0.20, and 0.70 +/- 0.23 pF, respectively. Antibodies against synaptotagmin I, a vesicle protein associated with fast exocytosis, strongly stained the synaptic terminal of isolated photoreceptors, suggesting the presence of fusion-competent vesicles. Our results confirm that photoreceptors possess a large rapidly releasable pool activated by a low-affinity Ca2+ sensor whose kinetic and calcium-dependent properties are similar to those reported in retinal bipolar cells and cochlear hair cells.

Animals↗
PubMed2002

Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+] in pituitary cells.

Synaptotagmin I (Syt I), a low-affinity Ca(2+)-binding protein, is thought to serve as the Ca(2+) sensor in the release of neurotransmitter. However, functional studies on the calyx of Held synapse revealed that the rapid release of neurotransmitter requires only approximately micromolar [Ca(2+)], suggesting that Syt I may play a more complex role in determining the high-affinity Ca(2+) dependence of exocytosis. Here we tested this hypothesis by studying pituitary cells, which possess high- and low-affinity Ca(2+)-dependent exocytic pathways and express Syt I. Using patch-clamp capacitance measurements to monitor secretion and the acute antisense deletion of Syt I from differentiated cells, we have shown that the rapid and the most Ca(2+)-sensitive pathway of exocytosis in rat melanotrophs requires Syt I. Furthermore, stimulation of the Ca(2+)-dependent exocytosis by cytosol dialysis with solutions containing 1 microM [Ca(2+)] was completely abolished in the absence of Syt I. Similar results were obtained by the preinjection of antibodies against the CAPS (Ca(2+)-dependent activator protein for secretion) protein. These results indicate that synaptotagmin I and CAPS proteins increase the probability of vesicle fusion at low cytosolic [Ca(2+)].

Animals↗
PubMed2000

Dopaminergic regulation of synaptotagmin I and IV mRNAs in hemiparkinsonian rats.

Synaptotagmins (Syts) I and IV are synaptic proteins involved in the regulation of neurosecretion. Dopaminergic drugs have been shown to modulate their expression. Here we investigate whether dopaminergic regulation of syt I and syt IV expression could play a role in the hypersensitive striatum of rats with unilateral lesions of dopaminergic nigrostriatal neurons with 6-hydroxydopamine. We show that chronic dopaminergic denervation resulted in a small down-regulation of striatal syt I mRNA, whereas acute treatment with SKF-82958, a dopamine D1 receptor agonist, induced a massive syt IV mRNA upregulation in the striatum on the lesioned side. We conclude that chronic lack of dopamine and treatment with dopamine D1 receptor agonists alter the synaptic plasticity in dopamine depleted basal ganglia.

Animals↗
PubMed2000

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein.

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca(2+)-dependent activator protein for secretion), a protein required for Ca(2+)-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca(2+) elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca(2+) dependencies. A threshold of approximately 10 microM Ca(2+) was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca(2+) activity < 10 microM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca(2+) and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Animals↗
PubMed2000

Transient and permanent fusion of vesicles in Zea mays coleoptile protoplasts measured in the cell-attached configuration.

Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (Cm). Vesicle fusion was observed as (i) An irreversible step increase in Cm. (ii) Occasionally, irreversible Cm steps were preceded by transient changes in Cm, suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in Cm did not lead to an irreversible Cm step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction.

Cell Membrane↗
PubMed2000

Actin cytoskeleton and exocytosis in rat melanotrophs.

We monitored secretory activity of single rat melanotrophs by the patch-clamp membrane capacitance measurements (Cm). Secretory activity was stimulated by cytosol dialysis with a patch-pipette solution containing 1 microM [Ca2+]i. Actin cytoskeleton was disaggregated by pretreating cells with Clostridium spiroforme toxin, which specifically ADP-ribosylates cellular actin. The extent of cytoskeleton disaggregation was monitored by phalloidin immunostaining. The maximal rate of secretion increases two folds in toxin-treated cells in comparison to controls, whereas the extent of calcium-induced secretory response was similar to that obtained in the non-treated cells. The results show that the subcortical actin network attenuates the rate of secretory activity, which we interpret to reflect a barrier function of cytoskeleton for exocytosis.

Actins↗
PubMed2000

Alpha-melanocyte stimulating hormone desensitizes the responsiveness of carbon-fibres within seconds.

We monitored electrooxidation of noradrenaline and alpha-melanocyte stimulating hormone (alpha-MSH) at a carbon-fibre microelectrode (CFME). The solution of noradrenaline (1 mM) or alpha-MSH (1 mM) was applied by a pressure pulse (2 s) from a micropipette to a voltage-clamped (850 mV) CFME immersed into bathing solution of an inverted microscope chamber. The distance between the CFME and micropipette was 2 to 12 microm. The maximal currents recorded for these two agents were 8.0 +/- 0.5 pA (N = 9) and 3.0 +/- 1.1 pA (N = 9), respectively. Pressure application of control solution did not affect the measured current. The noradrenaline-evoked anodic current was characterized by a monotonic increase that attained the maximum at the end of the pressure pulse. In contrast, the time-course of the alpha-MSH-evoked current was biphasic. The maximum amplitude of this current was attained in 0.59 +/- 0.15 s (N = 9) and then it declined with a time constant of 7.5 +/- 4.0 s (N = 9) until the pressure pulse was terminated. We explain this phenomenon to be due to an interaction between the peptide oxidation products and the CFME which results in its desensitization.

Carbon↗
PubMed1999

Actin cytoskeleton depolymerization with clostridium spiroforme toxin enhances the secretory activity of rat melanotrophs.

1. We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerizes cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 microM Ca2+i increased Cm by 23 +/- 4 % (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 +/- 5 % (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 +/- 0.85 fF s-1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s-1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P < 0.01). 3. In control cells cytosol dialysis with 0 microM Ca2+i decreased Cm by 9 +/- 3 % (n = 7), in CST-treated cells Cm increased by 11 +/- 3 % (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s-1; CST treatment: 1-2 fF s-1). 4. Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis.

ADP Ribose Transferases↗
PubMed1999

The heterotrimeric Gi(3) protein acts in slow but not in fast exocytosis of rat melanotrophs.

Besides having a role in signal transduction some trimeric G-proteins may be involved in a late stage of exocytosis. Using immunocytochemistry and confocal microscopy we found that Gi(3)-protein resides mainly in the plasma membrane, whereas Gi(1/2-)protein is preferentially associated with secretory granules. To study the function of trimeric Gi(3)- and Gi(1/2)-proteins, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. We report here that mastoparan, an activator of trimeric G-proteins, enhances calcium-induced secretory activity in rat melanotrophs. The introduction of synthetic peptides corresponding to the C-terminal domain of the (&agr;)-subunit of Gi(3)- and Gi(1/2)-proteins indicated that Gi(3 )peptide specifically blocked the mastoparan-stimulated secretory activity, which indicates an involvement of a trimeric Gi(3)-protein in mastoparan-stimulated secretory activity. Flash photolysis of caged Ca(2+)-elicited biphasic capacitance increases consisting of a fast and a slower component. Injection of anti-Gi(3) antibodies selectively inhibited the slow but not the fast component of secretory activity in rat melanotrophs. We propose that the plasma membrane-bound Gi(3)-protein may be involved in regulated secretion by specifically controlling the slower kinetic component of exocytosis.

Animals↗
PubMed1998

Modulation of the unitary exocytic event amplitude by cAMP in rat melanotrophs.

1. Secretory responses were measured in single rat pituitary melanotrophs as the relative increase in membrane capacitance (Cm) 8 min after the start of dialysis with solutions containing 0.45 microM Ca2+. In the added presence of cAMP (0.2 mM) in the patch pipette solution, capacitance responses increased 2- to 3-fold in comparison with controls. 2. To study whether cAMP-dependent mechanisms affect cytosolic calcium activity ([Ca2+]i), dibutyryl cyclic AMP (dbcAMP, 10 mM) was added to intact melanotrophs and [Ca2+]i was measured using fura-2 AM. Addition of dbcAMP caused a transient reduction in [Ca2+]i to 82 +/- 21 nM from a resting value of 100 +/- 19 nM (mean +/- S.E.M., n = 32, P < 0.002), indicating that the cAMP-induced increase in secretory activity was not the result of cAMP acting to increase [Ca2+]i, which then increased secretory activity. 3. To investigate whether cAMP affects the secretory apparatus directly, the interaction of a single secretory granule with the plasmalemma was monitored by measuring discrete femtofarad steps in Cm. The signal-to-noise ratio of recordings was increased by pre-incubating the cells with a hydrophobic anion, dipicrylamine. 4. Recordings of unitary exocytic events (discrete 'on' steps in Cm) showed that the amplitude of 'on' steps - a parameter correlated to the size of exocytosing secretory granules - increased from 4.2 +/- 0.2 fF (n = 356) in controls to 7.9 +/- 0.2 fF in the presence of cAMP (n = 329, P < 0.001), while the frequency of unitary exocytic events was similar in controls and in the presence of cAMP. 5. The results suggest that a cAMP-dependent mechanism mediates the fusion of larger granules with the plasmalemma.

Adenosine Triphosphate↗
PubMed1997

Mastoparan and Rab3AL peptide potentiation of calcium-independent secretory activity in rat melanotrophs is inhibited by GDPbetaS.

The whole-cell patch-clamp membrane capacitance measurement was used to monitor secretory activity in rat melanotrophs, while rab3AL, putative effector domain peptides of Rab3 small GTPases (20-30 kDa), were introduced into cytosol. In melanotrophs dialyzed with calcium free solutions membrane capacitance tends to decrease slightly. This decrease is further potentiated with GDPbetaS (500 microM). We found that rab3AL (100 microM) stimulated secretory activity in the absence of calcium. The rab3AL response was qualitatively comparable to the response to mastoparan (1 microM), an activator of certain heterotrimeric GTP-binding proteins. Interestingly, inclusion of GDPbetaS (500 microM) resulted in a blockade of both rab3AL and mastoparan induced responses. We conclude that rab3AL and mastoparan induce calcium-independent stimulation of secretory activity in rat melanotrophs by activation of a downstream heterotrimeric GTP-binding protein.

Amino Acid Sequence↗
PubMed1997

Cell-attached measurements of attofarad capacitance steps in rat melanotrophs.

Capacitance changes in cell-attached patches of rat melanotrophs were measured by a high-frequency lock-in amplifier. The background noise of around 30 aF allowed the detection of discrete steps due to fission (endocytosis) and fusion (exocytosis) of vesicles with diameters as small as 60 nm. The amplitude of both types of steps was similar with a modal value of around 300 aF. The frequency of these steps was not changed, if secretagougues such as ionomycin and/or dibutyril cAMP, were applied to the bathing solution. Moreover, this treatment did not result in an increased appearance of expected 2000 to 3000 aF steps due to exocytosis of secretory granules. We conclude that the likely explanation for recorded capacitance steps is that they represent the constitutive vesicle traffic. From the typical frequency and amplitude of these events (around 1 min-1, 300 aF) in a membrane patch (26 fF) it is estimated that the whole membrane of a rat melanotroph may be ingested under our conditions in 1 to 2 h.

Animals↗
PubMed1996

Nicardipine enantiomers inhibit calcium and outward currents in rat pars intermedia cells.

The whole-cell patch clamp technique was used to investigate the effect of the dihydropyridine antagonist nicardipine and its two enantiomers on calcium and outward currents in rat pars intermedia cells. Inward voltage-activated calcium currents as well as outward currents were blocked by nicardipines (enantiomers and racemic substance). We found a highly significant correlation between the blockage of voltage-activated currents and outward currents, indicating an association between recorded currents. Therefore, the inhibitory effect of nicardipines was measured as outward current inhibition. The effects of (-) and (+) enantiomers as well as the racemic nicardipine were concentration-dependent. The (+)-enantiomer was the least potent with an ED50 of 3.8 +/- 1.2 microM, while the (-)-enantiomer inhibited currents with an ED50 of 0.7 +/- 0.1 microM. Racemic nicardipine blocked currents with an ED50 of 2.2 +/- 0.6 microM.

Animals↗
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↗