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CARDIOVASCULAR

Single-Vesicle Catecholamine Release Has Greater Quantal Content and Faster Kinetics in Chromaffin Cells from Hypertensive, as Compared with Normotensive, Rats

Instituto Teófilo Hernando para la Investigación de Fármacos y del Envejecimiento, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain (R.M.-F., R.d.P., A.M.G.d.D., L.G., A.G.G.); Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, Brazil (R.M.-F., A.C.-N., A.J.); and Servicio de Farmacología Clínica, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain (A.G.G.)

Received July 18, 2007; accepted October 24, 2007.

	Abstract

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In a previous study performed in the intact adrenal gland (Lim et al., 2002), stimulation with acetylcholine (ACh) or high K⁺ concentrations (K⁺) produced greater catecholamine release in spontaneously hypertensive rats (SHR), as compared with normotensive animals. In this study, the time course of secretion was in the range of minutes. Hence, we do not know whether enhanced release is due to greater quantal content and/or distinct kinetics in SHRs and control animals. To get insight into the mechanism involved in such enhanced catecholamine secretory responses, we performed a single-vesicle release study in primary cultures of adrenal chromaffin cells, recorded with amperometry. Cells were stimulated with 2-s pulses of 1 mM ACh or 70 mM K⁺. The secretory responses to ACh or K⁺ pulses in SHR cells as compared with control cells had the following characteristics: 1) double number of secretory events, 2) 4-fold augmentation of total secretion, 3) cumulative secretion that saturated slowly, 4) 3-fold higher complex events with two to four superimposed spikes that may be explained by faster spike kinetics, 5) about 2- to 3-fold higher event frequency at earlier post stimulation periods, and 6) 2- to 5-fold higher quantal content of simple spikes. We conclude that SHR cells have faster and larger catecholamine release responses, explained by more vesicles ready to undergo exocytosis and greater quantal content of vesicles. This could have relevance to further understand the pathogenic mechanisms involved in the development of high blood pressure, as well as in the identification of new drug targets to treat hypertension.

Numerous studies performed with different methodologies support that pre- and postsynaptic sympathetic dysfunctions are involved in the pathophysiology of primary hypertension in humans or laboratory animals (Tsuda and Masuyama, 1991; de Champlain et al., 1999). Studies on presynaptic mechanisms have been performed in the SHR, a model of primary hypertension; they show that norepinephrine release is increased in different tissues rich in sympathetic nerve endings (Donohue et al., 1988). Although this increase of NA release from sympathetic nerve endings constitutes an important catecholaminergic dysfunction associated to primary hypertension, the precise mechanism involved in this dysfunction remains unknown.

A recent study on catecholamine release from intact perfused rat adrenal glands indicates that release stimulated by acetylcholine (ACh), the physiological neurotransmitter at the adrenal medulla splanchnic nerve-chromaffin cell synapse (Feldberg et al., 1934), or by high K⁺ concentrations (K⁺), is higher in adrenals from SHR, as compared with the responses obtained in control normotensive rats (Lim et al., 2002). However, in this study, secretion was assessed upon collection of 4-min perfusate samples, with a poor temporal resolution to study a fast secretory event such as exocytosis of chromaffin cells (Neher, 1998). This low-temporal resolution precluded the analysis in such study of the fast kinetics of secretion or quantal aspects of single-vesicle secretory events upon stimulation of isolated individual chromaffin cells from control and SHR animals.

Thus, we felt that a study to analyze the kinetics of single-vesicle secretory events, using a carbon fiber electrode and amperometry, was timely. This high-resolution technique provides insight not only on quantitative, but also on qualitative, kinetic aspects of the last fusion steps of exocytosis in isolated chromaffin cells in the millisecond time range (Wightman et al., 1991; Borges et al., 2005). We found that short pulses (2 s) of ACh or K⁺ elicited a more sustained production of spike secretory events and a drastic augmentation of the quantal catecholamine content of individual secretory vesicles, which had faster fusion kinetics in SHR, as compared with normotensive rats.

	Materials and Methods

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Control and Spontaneously Hypertensive Rats. Animals were manipulated according to the guidance of the Ethics Committee for Handling Research Animals of our medical school, Universidad Autónoma de Madrid. Male 16-week-old Sprague-Dawley and SHR weighing around 300 g were housed at 24 ± 2°C with 60 ± 20% relative humidity on a 12-h light/12-h dark cycle. Animals were fed a standard diet and water ad libitum and periodically weighed. Systolic blood pressure, diastolic blood pressure, and heart rate (beats per minute) were evaluated with a pressure meter (LETICA 2006; Cibertec, Madrid, Spain) by cannulating the femoral artery. As in our present study, several other studies on hypertensive rats used Sprague-Dawley rats as control normotensive rats (Wexler, 1980; Chao and Chao, 1988; Jandeleit-Dahm et al., 1997; Shimosawa et al., 2004).

Isolation and Culture of Rat Adrenal Medulla Chromaffin Cells from Control and Hypertensive Rats. To prepare each cell batch, we used one to two adult rats that were killed by cervical dislocation. The abdomen was opened, the adrenal glands were exposed and quickly removed and decapsulated, and both adrenal medullae were isolated under a stereoscope. They were placed in Ca²⁺- and Mg²⁺-free Locke buffer of the following composition: 154 mM NaCl, 3.6 mM KCl, 5.6 mM NaHCO₃, 5.6 mM glucose, and 10 mM Hepes, pH 7.2, at room temperature. Tissues were collected under sterile conditions. Medullae digestion was achieved by incubating the pieces in 6 ml of Ca²⁺/Mg²⁺-free Locke buffer containing 6 mg of collagenase and 12 mg of bovine serum albumin for 20 min at 37°C; gentle agitation was applied at 5- to 10-min intervals by using a plastic Pasteur pipette. The collagenase was washed out of the cells with large volumes of Ca²⁺/Mg²⁺-free Locke buffer. The cell suspension was centrifuged at 120g for 10 min. After washing two times, the cells were resuspended in 1 ml of Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum containing 50 IU/ml penicillin and 50 µg/ml streptomycin.

Cells were plated on circular glass coverslips, previously treated with 0.1 mg/ml poly-D-lysine for 30 min, followed by a thorough washout with water. After 30 min, 1 ml of DMEM was added to each well. Cells were then incubated at 37°C in a water saturated, 5% CO₂ atmosphere; they were used within 1 to 2 days after plating.

Amperometric Monitoring of Catecholamine Release with a Carbon Fiber Microelectrode. Carbon fiber microelectrodes were prepared by cannulating a 7-µm-diameter carbon fiber in polyethylene tubing. The carbon fiber tip was glued into a glass capillary for mounting on a patch-clamp head stage and backfilled with 3 M KCl to connect to the Ag/AgCl wire, which was kept at +700 mV. The electrode was positioned at the middle right side of a spherical cell, gently touching the cell. Amperometric currents were recorded using an EPC-9 amplifier and PULSE software running on an Apple Macintosh computer (Apple Computer, Cupertino, CA). Sampling was performed at 14.5 kHz, and samples were digitally filtered at 2 kHz. The sensitivity of the electrodes was routinely monitored before and after the experiments, using 50 µM epinephrine as standard solution. Only fibers that rendered 200 to 300 pA of current increment after a 50 µM epinephrine pulse were used for the experiments. Cell secretion was stimulated by pulses of 70 mM K⁺ or 1 mM ACh for 2 s, delivered from a micropipette located 40 µm away from the cell right side of the cell being explored; solutions bathed the cells by gravity, upon opening of computer-driven valves.

Spike Analysis and Statistics. Spike analysis was performed using the Pulse Program (HEKA, Lambrecht/Pfalz, Germany) and Igor Pro Software (Max Planck Institute, München, Germany), which includes the Ricardo Borges's macro package that allows the analysis of single events (Segura et al., 2000). A threshold of 4.5 times the first derivative of the noise S.D. was calculated to clearly detect amperometric events.

Differences between means of group data fitting a normal distribution were assessed by using Student's t test. A p value equal or smaller than 0.05 was taken as the limit of significance. The cumulative secretion (Figs. 1C and 2C) was analyzed by repeated measures with ANOVA followed by the Bonferroni test.

View larger version (17K): [in this window] [in a new window]   Fig. 1. ACh pulses produced longer lasting catecholamine secretory responses in SHR chromaffin cells, compared with control cells. A, example record obtained from a control cell stimulated with an ACh pulse (1 mM ACh, 2 s), as indicated by the horizontal bar at the bottom. B, record obtained from an example SHR cell, similarly stimulated with an ACh pulse. Note the prolonged duration of the period showing poststimulus amperometric spikes. C, cumulative secretion, calculated at 2-s intervals in traces similar to those shown in A and B; the area of spikes is expressed and represented in pC (ordinate) as a function of time after the ACh pulse (abscissa). Data are means ± S.E. of the number of cells shown in parentheses. Statistical difference between control and SHR cells was at p < 0.001 (***), using Bonferroni test after analysis by repeated measures with ANOVA.

View larger version (18K): [in this window] [in a new window]   Fig. 2. High-K+ pulses produced longer lasting catecholamine secretory responses in SHR chromaffin cells, as compared with control cells. A, example record obtained from a control cell stimulated with a high-K+ pulse (70 mM K+, 2 s), as indicated by the horizontal bar at the bottom. B, record obtained from an example SHR cell, similarly stimulated with K+. Note the striking poststimulus secretory spike activity in this SHR cell. C, cumulative secretion, calculated at 2-s intervals from traces similar to those of A and B and represented in pC (ordinate) as a function of time. Data are means ± S.E. ***, p < 0.001, ANOVA.

	Results

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Body Weight, Adrenal Weight, and Hemodynamic Parameters of Control and SHR. Sixteen-week-old rats were used. Control rats weighed 328.2 g, and SHR weighed 318.9 g (Table 1). The dry weights of adrenal glands were 22.3 mg for control and 22.7 mg for SHR. The hemodynamic parameters were significantly higher in SHR as shown in Table 1. Thus, the systolic blood pressure, the diastolic blood pressure, and the mean arterial pressure were 55.2, 59.3, and 56.4% higher than the values obtained in control animals. The basal heart rate of control rats was 337 bpm and that of SHR animals 371 bpm, 10.2% higher.

The Amperometric Catecholamine Secretory Responses Elicited by ACh. Occasionally, rat adrenal chromaffin cells maintained in primary cultures fire low-frequency spontaneous action potentials (Kidokoro et al., 1982). This predicts that cells might spontaneously produce some secretory spikes. However, in our experiments, this spontaneous activity was rare, and when produced, the cell was discarded.

Usually, an experiment began with about 2- to 5 min of cell perifusion with the basal Krebs-Hepes solution to allow its equilibration. When possible, two or more cells were explored with the carbon fiber microelectrode in the same coverslip, going from right (the position of the microelectrode) to left (the position of the local superfusion pipette). In this manner, we did not expose to ACh or K⁺ stimuli the second or third cell explored in the same coverslip.

Figure 1A shows a prototype secretory response elicited by an ACh pulse (1 mM, given focally for 2 s) in a control cell. After a delay of about 600 ms, a burst of secretory spikes appeared. The duration of the burst overlasted the ACh application by about 2.5 s. The spike frequency was initially higher to decline after 2 s or so. Note that the rest of the trace was silent, and no spikes clearly different from noise were produced.

The behavior of the SHR cell shown in the trace of Fig. 1B was quite different. First, the delay between the ACh pulse and the appearance of the first spike was considerably shortened to about 150 ms. Second, the baseline secretion was markedly enhanced likely as a result of overlapping secretory spikes. Third, the amplitudes of some spikes seemed to be larger. Fourth, the spike response overlasted the ACh pulse duration during a substantially longer period, about 15 s.

Figure 1C shows a plot of the mean cumulative secretion from various cells explored following the protocol of Fig. 1, A and B. The secretion evoked by the ACh pulse in each individual cell was calculated at 2-s intervals because the area of spikes present within such 2-s period that was expressed in picoculombs (pC) of catecholamine released and cumulatively added to obtain the curves of Fig. 1C. Note that in control cells, secretion during the first 6 s after the ACh pulse saturated at about 120 pC and stopped after 14 s. In contrast, in SHR cell secretion continued rising to about a 600 pC peak at the 35th s.

The Amperometric Catecholamine Secretory Responses Elicited by K⁺. Saline solutions containing high K⁺ concentrations cause chromaffin cell depolarization, enhanced Ca²⁺ entry, and catecholamine release. This stimulus directly recruits voltage-dependent Ca²⁺ channels, thus bypassing the nicotinic receptors stimulated by ACh (Garcia et al., 2006). Hence, to define whether ACh evoked greater responses in SHR cells just through a nAChR-delimited effect, secretion was also studied in cells stimulated with K⁺-depolarizing saline solutions.

In the experiments shown in Fig. 2A, a protocol similar to that of Fig. 1 was followed, except for the fact that here, a control cell was stimulated for 2 s with a solution containing 70 mM K⁺ (isoosmolar reduction of Na⁺). Note that the secretory spikes appeared soon after K⁺ application, with a delay of only 200 ms; the secretory activity overlasted the K⁺ pulse and kept going for an additional 4-s period.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Decay of secretory responses upon repeated cell pulsing with ACh (1 mM, 2 s) or K+ (70 mM, 2 s) is much more pronounced in SHR cells, as compared with control cells. A, total secretion per pulse (integrated area of all spikes obtained in each stimulus, in pC, ordinate) in control and SHR cells stimulated twice with a 5-min interval. B, total number of spikes secreted per each ACh pulse in the same cells of A. C and D, total secretion and spike number per pulse, respectively, in cells stimulated with three successive K+ pulses given at 5-min intervals. Data are means ± S.E. of the number of cells given in parentheses. *, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to control cells (Student's t test).

Decay of Secretory Responses upon Repeated Pulsing with ACh or K⁺. In a few SHRs and in most control cells, repeated pulsing with ACh or K⁺ produced secretory responses that decayed with time. For instance, in four SHR cells, we could obtain two responses evoked by ACh pulses separated by a 5-min interval. Figure 3A shows that the second secretory responses decayed to about 50% in control cells and by 40% in SHR cells. However, the responses to both stimuli were significantly higher in SHR cells, about 2.5-fold above control cells. The number of spikes also decreased during the second ACh pulse in both cell types, but once more, it was higher in SHR cells.

Secretory responses also decayed with successive high-K⁺ pulses (Fig. 3C). For instance, in three SHR cells, the total secretion per pulse (Q) decreased from 220 pC (first pulse) to about 100 pC (second pulse) and to around 60 pC (third pulse). A similar relative decline was seen in control cells, although such decline was smaller from the second to the third K⁺ stimulus. Again, secretion was 3- to 4-fold higher in SHR cells, as compared with control cells during the first and second stimuli. A similar picture was observed on the total number of spikes per K⁺ pulse; spike numbers were higher in SHR cells during the first and second K⁺ stimuli, whereas they were similar to control cells during the third stimulus. Since the decay of secretion to repeated pulsing was high, particularly in SHR cells, we performed the kinetic analysis of single-vesicle events by taking into account only the secretory responses elicited by each ACh or K⁺ pulse in every individual cell.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Incidence of single and multiple amperometric spike events in the secretory responses evoked by ACh (1 mM, 2 s) or high K+ pulses (70 mM K+, 2 s), in control and SHR cells. A and B, examples of single spikes, with or without a foot. C and D, samples of multiple spikes. To draw the histogram of E, single or multiple spikes were counted in the trace secretory responses elicited by ACh or K+. The incidence of multiple spikes in each individual cell was expressed as percentage of total spikes, counting single or "multiple" spikes as "individual" events. Data are means ± S.E. ***, p < 0.001 with respect to control (Student's t test).

We counted the number of single or multiple spikes in all traces recorded from control and SHR cells stimulated with ACh or K⁺ pulses (Fig. 4E). In control cells stimulated with ACh, 10% of the 599 spikes analyzed had a complex profile. In SHR cells, 30% of spikes were complex, of the 834 spikes analyzed. A similar picture emerged upon the analysis of K⁺-elicited spikes; multiple spikes accounted for 9% in control cells and were 3.5-fold more frequent in SHR cells.

Frequency Distribution of Secretory Events in the Responses Elicited by ACh or K⁺ Pulses. Another facet of interest was the analysis of spike frequency along the traces obtained in the example cells shown in Figs. 1 and 2. Such analysis was performed second by second and is presented in Fig. 5A, showing the responses elicited by ACh. During the first 9 s of the trace, the spike frequency was in general higher in SHR, compared with control cells. From this time to the 27th s, the control cell was silent, whereas the SHR cell continued discharging spike events at about 5 to 7 Hz.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Histograms of frequency spike distribution, calculated from the traces shown in Figs. 1, A and B, and 2, A and B, corresponding to the secretory responses elicited by ACh or K+, in control and SHR cells. The number of spikes present in 1-s time periods were calculated and plotted as frequencies in Hz (ordinates). Data are representative of cells stimulated with a 2-s ACh pulse (as indicated by the bottom horizontal bar in A) or by a 2-s K+ pulse (as shown by the horizontal bar in B).

The responses to K⁺ were considerably faster during the first 10 s, as indicated in Fig. 5B. For instance, during the first 5 s, the control spikes reached frequencies between 7 to 17 Hz (compared with about 10 Hz with ACh); the spikes from SHR cells reached frequencies as high as 40 Hz.

Frequency Distribution of Secretory Events as a Function of Spike Amplitude. A significant higher spike number was counted along the secretory responses elicited by ACh or K⁺ in SHR cells, with respect to control cells. The question is whether the distribution of spikes was uniform at different spike amplitudes, or rather, if they were randomly distributed along the trace recording obtained with each stimulus, independently of amplitudes.

Figure 6A shows the number of spikes generated by ACh pulsing as a function of amplitude ranges, grouped in 200-pA steps. In the lower amplitude range (0–199 pA), 39 spikes were found in control cells and 76 spikes in SHR cells, about 2-fold higher. In the range of 200 to 399 pA, these values decreased to 9 and 14 spikes, respectively. The spikes with higher amplitudes were few and had no statistical differences between control and SHR.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Distribution of secretory spikes as a function of their amplitudes, in control and SHR cells stimulated with ACh or K+ pulses. Spikes from all traces obtained in the experiments shown in Figs. 1 and 2 were grouped according to their amplitudes in the 200-pA range (abscissae), counted, and their numbers are given in the ordinates. A, spike distribution in cells stimulated with ACh pulses; B, that obtained from cells stimulated with K+ pulses. Data are means ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001, with respect to control cells (Student's t test).

In the case of spikes elicited by K⁺, a similar pattern emerged. So, in the lower range (0–199 pA), control cells exhibited 54 spikes, whereas SHR cells had 103 spikes, about 2-fold higher. In the range of 200 to 399 pA, nine spikes were in control cells and 19 spikes in SHR cells. Again, at higher amplitudes, fewer spikes were seen.

Catecholamine Quantal Content and Kinetics of Single Amperometric Spikes. We also analyzed the quantal catecholamine content of individual spikes (Q, in pC). Partially superimposed spikes that begun above baseline or that did not reach baseline when decaying were hard to accurately estimate their area and hence their quantal catecholamine contents; therefore, we did not include them in this analysis. The area of these single spikes reflects the release of the total vesicular content of catecholamines (Zhou et al., 1996).

Spikes were distributed in groups according to their amplitudes, in 200-pA steps. The maximal numbers of spikes were found in the range of 0- to 199-pA amplitude, both for ACh and K⁺ pulses, in control as well as SHR cells (Fig. 7,A and B). In this range, the quantal content of spikes from control cells stimulated with ACh approached 1 pC, in agreement with Q values of 0.8 pC of mouse chromaffin cells (Arroyo et al., 2006) and 1.3 pC in bovine chromaffin cells (Ardiles et al., 2007). A surprising finding was the much higher Q of SHR spikes, about 3- to 5-fold higher than the values of Q for control cells stimulated with ACh. The spikes triggered by K⁺ exhibited a pattern similar to ACh (Fig. 7B); their quantal content was about 2- to 4-fold higher in SHRs, compared with control cells.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Analysis of the quantal content of individual secretory events obtained from control and SHR cells (Figs. 1 and 2). Spike areas (quantal catecholamine content of individual vesicles, expressed in pC, Q, in the ordinates) were individually analyzed and distributed according to the amplitude range, in pA, shown in the abscissae. Data are means ± S.E. of the number of spikes shown in parentheses on top of each column. A, analysis of spikes obtained with ACh stimulation of control and SHR cells. B, corresponding to K+ stimulation. Data are means ± S.E. of the number of spikes shown in parentheses on top of each column. **, p < 0.01; ***, p < 0.001, with respect to control cells.

We also analyzed the kinetics of individual spikes. Table 2 shows that the time to peak (t_max) was around 16 ms in spike events elicited by ACh or K⁺ stimulation of control cells; t_max was reduced by about 40% in SHR spikes. This indicated faster spike ascension (m) that was 35 to 50% higher in SHR cells, compared with control cells. The spike half-width (t_1/2) was reduced by 21% in SHR cells.

	Discussion

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We have found that the stimulation of single rat adrenal chromaffin cells with short ACh or K⁺ pulses causes catecholamine secretory responses that are substantially higher and longer lasting in cells obtained from SHRs, as compared with responses generated in cells from control normotensive animals (Figs. 1, 2, 3). This corroborates the observation of Lim et al. (2002), who in the perfused intact adrenal gland observed that stimulation with ACh or K⁺ enhanced more the catecholamine present in 4-min perfusate samples of SHRs, as compared with control rats. However, in such a study using the intact gland, the slow perfusion rate and the low sensitivity of the catecholamine fluorescence assay permitted only the analysis of global catecholamine release responses in the minute range, precluding to get insight into the kinetic mechanisms involved in such enhanced secretion. In our study, we measured the amperometric detection of single-vesicle exocytotic events in cultured single chromaffin cells, allowing us a high temporal resolution (milliseconds to seconds) analysis of secretory responses. We found that the integrated total catecholamine release elicited by an ACh pulse was 4-fold higher in SHRs, as compared with control rats (Fig. 1C). However, the number of secretion spikes generated by an ACh pulse was only 2-fold higher in SHR cells (Fig. 1D). In cells stimulated with K⁺ pulses, a similar outcome was seen (Fig. 2, C and D). Thus, augmented secretion in SHRs versus control cells may have at least two components: 1) a higher number of secretory events and 2) a higher quantal content of individual events, as shown in Fig. 7.

Another interesting feature was related to the number of events having single or multiple spikes. It is generally accepted that a separate well identified spike corresponds to the quantal total release of the catecholamine stored in a single secretory vesicle, through a fusion pore formed by the fusion of the vesicle membrane with the plasmalemma (Breckenridge and Almers, 1987); this pore usually expands to release the total vesicular content expressed by the area of a single spike (Zhou et al., 1996). Multiple spike events indicate that various vesicles suffered almost simultaneous exocytosis; this was more pronounced with K⁺ stimulation of SHR cells, which caused a clear elevation of baseline secretion (Fig. 2B). In SHR cells, about 30% of events had multiple spikes (two to four spikes), whereas control cells had only 10% of events with multiple spikes; this was true with ACh or K⁺ stimulation (Fig. 4E), suggesting that in SHR cells, more vesicles were docked and primed at subplasmalemmal sites (Neher, 1998) to undergo exocytosis upon ACh or K⁺ stimulation. Alternatively, it may also indicate that vesicle fusion with the plasmalemma and the ensuing quantal catecholamine release was faster (Table 2), giving rise to more exocytotic sites available and to more spike overlapping.

It is unlikely that differences in the nicotinic receptors and/or voltage-dependent Ca²⁺ channels present in rat chromaffin cells (Garcia et al., 2006) could explain the different secretion pattern seen in SHRs, as compared with control cells. This conclusion is supported by the quite similar responses generated by ACh, which indirectly depolarizes the chromaffin cells (Douglas et al., 1967) and activates exocytosis by enhancing Ca²⁺ entry through Ca²⁺ channels (Douglas and Poisner, 1961) or by high K⁺ concentrations that cause direct cell depolarization (Douglas et al., 1967) and recruitment of voltage-dependent Ca²⁺ channels in both control and SHR cells. However, it will be interesting to study the inward currents through nicotinic receptors generated by ACh, as well as the inward Ca²⁺ currents generated by square depolarizing pulses or by action potentials in both normotensive and hypertensive rats.

Another possibility rests in a different Ca²⁺ homeostatic mechanism in SHRs as compared with control cells. We have seen different patterns of exocytosis in bovine and mouse chromaffin cells, likely related to different Ca²⁺ handling by mitochondria, upon cell depolarization with K⁺ or electrically evoked depolarizing pulses (Ales et al., 2005). Also, we found differences in ACh- or K⁺-evoked secretory responses in bovine chromaffin cell populations when the endoplasmic reticulum Ca²⁺ movements were disturbed (Cuchillo-Ibanez et al., 2002). A functional triad controlling [Ca²⁺]_c and exocytotic signals has been described to be present in bovine chromaffin cells using endoplasmic reticulum- or mitochondria-targeted aequorins and measurements of inward Ca²⁺ channel currents (Alonso et al., 1999; Montero et al., 2000; Garcia et al., 2006). It will be interesting to test whether such functional triad is present in chromaffin cells of control normotensive rats and if it is altered at some points in hypertensive rats. Alterations of these Ca²⁺ homeostatic mechanisms occur in vascular smooth muscle cells. In fact, a large Ca²⁺ influx and a high [Ca²⁺]_c are reported even during rest in conduit arteries (e.g., the aorta and the carotid and femoral arteries) from SHRs as compared with normotensive rats (Jelicks and Gupta, 1990; Asano et al., 1996); this has also been found in small mesenteric arteries from SHRs, where the sarcoplasmic reticulum has a larger capacity for Ca²⁺ storage (Nomura and Asano, 2002).

In conclusion, we have shown that short ACh or K⁺ pulses cause catecholamine secretory responses that drastically differ in chromaffin cells of normotensive and hypertensive rats. The much greater catecholamine release responses in SHRs are explained by the faster exocytosis of more vesicles with greater quantal catecholamine content, during a much longer secretory period. This drastic different behavior of the chromaffin cell secretory responses may contribute to the understanding of the pathogenic mechanism of hypertension development in humans and facilitate the identification of novel therapeutic targets to control high blood pressure.

	Acknowledgements

 
We thank Fundación Teófilo Hernando for continued support.

	Footnotes

 
This study and R.M.-F. were supported by a collaborative exchange research grant between the Governments of Spain and Brazil (PHB 2005-0018-PC), by the Departamento de Farmacología, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Brazil (to A.J.), and by the Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain (to A.G.G.). This study was also supported by the Plan Nacional de Investigación, Ministerio de Educación y Ciencia, Spain (Grants SAF 2006-03589 to A.G.G. and SAF2004-07307 to L.G.) and by Redes Temáticas de Investigacíon Cooperativa Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (Grant RD 06/0026 to A.G.G.). R.M. was partially supported by the Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.128819.

ABBREVIATIONS: SHR, spontaneously hypertensive rat; ACh, acetylcholine; DMEM, Dulbecco's modified Eagle's medium; ANOVA, analysis of variance; pC, picoculomb(s).

Address correspondence to: Dr. Antonio G. García, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4. 28029 Madrid, Spain. E-mail: agg{at}uam.es

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