Biomedical subjects
R Zorec
Publications and source records attributed to R Zorec.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Control of exocytosis in a plant secretory cell by cytosolic Ca2+ and hydrostatic pressure.
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New approaches in the study of stimulus-secretion coupling in anterior pituitary cells.
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