Isolated Chromaffin Cells Research Articles

Untapped Potential: Real-Time Measurements of Opioid Exocytosis at Single Cells.

The endogenous opioid system is commonly targeted in pain treatment, but the fundamental nature of neuropeptide release remains poorly understood due to a lack of methods for direct detection of specific opioid neuropeptides in situ. These peptides are concentrated in, and released from, large dense-core vesicles in chromaffin cells. Although catecholamine release from these neuroendocrine cells is well characterized, the direct quantification of opioid peptide exocytosis events has not previously been achieved. In this work, a planar carbon-fiber microelectrode served as a "postsynaptic" sensor for probing catecholamine and neuropeptide release dynamics via amperometric monitoring. A constant potential of 500 mV was employed for quantification of catecholamine release, and a higher potential of 1000 mV was used to drive oxidation of tyrosine, the N-terminal amino acid in the opioid neuropeptides released from chromaffin cells. By discriminating the results collected at the two potentials, the data reveal unique kinetics for these two neurochemical classes at the single-vesicle level. The amplitude of the peptidergic signals decreased with repeat stimulation, as the halfwidth of these signals simultaneously increased. By contrast, the amplitude of catecholamine release events increased with repeat stimulation, but the halfwidth of each event did not vary. The chromogranin dense core was identified as an important mechanistic handle by which separate classes of transmitter can be kinetically modulated when released from the same population of vesicles. Overall, the data provide unprecedented insight into key differences between catecholamine and opioid neuropeptide release from isolated chromaffin cells.

Online Detection of Catecholamine Release from the Perfused Rat Adrenal Gland.

Retrogradely perfused adrenal glands have historically served for establishing many of our current knowledge on the stimulus-secretion coupling process. Although the use of intact adrenals has largely been switched to isolated chromaffin cells, adrenal glands are still a very valuable tool to characterize physiological and pharmacological questions. Even more, this is an excellent preparation for studying the splanchnic nerve/chromaffin cell interaction. In this chapter, we will provide the ways to (i) perform retrograde perfusion of isolated rat adrenals, (ii) the method to apply electrical splanchnic nerve stimulation, and (iii) the preparation of adrenals to conduct online electrochemical detection of catecholamine release.

The β-Hydroxybutyrate-GPR109A Receptor Regulates Fasting-induced Plasticity in the Mouse Adrenal Medulla.

During fasting, increased sympathoadrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of agouti-related peptide (AgRP), a peptidergic cotransmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild-type animals, 24-hour fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, reverse transcription polymerase chain reaction experiments confirmed the mouse adrenal medulla contains GPR109A messenger RNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to coordinate the homeostatic response to food deprivation.

RNA-seq of Isolated Chromaffin Cells Highlights the Role of Sex-Linked and Imprinted Genes in Adrenal Medulla Development

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.

Fasting evokes a homeostatic response that maintains circulating levels of energy-rich metabolites and increases the drive to eat. Centrally, this reflex activates a small population of hypothalamic neurons that are characterized by the expression of AgRP, a neuropeptide with an extremely restricted distribution. Apart from the hypothalamus, the only other site with substantial expression is the adrenal gland, but there is disagreement about which cells synthesize AgRP. Using immunohistochemistry, flow cytometry, and reverse transcription-polymerase chain reaction, we show AgRP is present in the mouse adrenal medulla and is expressed by neuroendocrine chromaffin cells that also synthesize the catecholamines and neuropeptide Y. Short-term fasting led to an increase in adrenal AgRP expression. Because AgRP can act as an antagonist at MC3/4 receptors, we tested whether melanotan II, an MC3/4 receptor agonist, could regulate pre- and postsynaptic signaling within the adrenal medulla. Melanotan II decreased the paired-pulse ratio of evoked synaptic currents recorded in chromaffin cells; this effect was blocked by exogenous AgRP. In contrast, neither melanotan II nor AgRP altered the optogenetically evoked release of catecholamines from isolated chromaffin cells. These results are consistent with the idea that AgRP regulates the strength of the sympathetic input by modulation of presynaptic MC3/4 receptors located on preganglionic neurons. We conclude that a small population of neuroendocrine cells in the adrenal medulla, and the arcuate nucleus of the hypothalamus, express AgRP and neuropeptide Y and are functionally involved in the systemic response to fasting.

Identification of muscarinic receptor subtypes involved in catecholamine secretion in adrenal medullary chromaffin cells by genetic deletion.

Activation of muscarinic receptors results in catecholamine secretion in adrenal chromaffin cells in many mammals, and muscarinic receptors partly mediate synaptic transmission from the splanchnic nerve, at least in guinea pigs. To elucidate the physiological functions of muscarinic receptors in chromaffin cells, it is necessary to identify the muscarinic receptor subtypes involved in excitation. To identify muscarinic receptors, pharmacological tools and strains of mice where one or several muscarinic receptor subtypes were genetically deleted were used. Cellular responses to muscarinic stimulation in isolated chromaffin cells were studied with the patch clamp technique and amperometry. Muscarinic M₁, M₄ and M₅ receptors were immunologically detected in mouse chromaffin cells, and these receptors disappeared after the appropriate gene deletion. Mouse cells secreted catecholamines in response to muscarinic agonists, angiotensin II and a decrease in external pH. Genetic deletion of M₁, but not M₃, M₄ or M₅, receptors in mice abolished secretion in response to muscarine, but not to other stimuli. The muscarine-induced secretion was suppressed by MT7, a snake peptide toxin specific for M₁ receptors. Similarly, muscarine failed to induce an inward current in the presence of MT7 in mouse and rat chromaffin cells. The binding affinity of VU0255035 for the inhibition of muscarine-induced currents agreed with that for the M₁ receptor. Based upon the effects of genetic deletion of muscarinic receptors and MT7, it is concluded that the M₁ receptor alone is responsible for muscarine-induced catecholamine secretion.

The Role of Doc2B in Depolarization-Evoked and G Protein-Coupled Receptor Modulated Exocytosis in Mouse Chromaffin Cells

Previously we demonstrated that activation of Gq protein-coupled H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine chromaffin cells, despite inhibiting Ca2+ influx through voltage-gated calcium channels (VGCCs). This potentiation is restricted to the immediately releasable pool (IRP) of vesicles coupled to VGCCs, and requires the priming protein Munc13-1. Doc2B is a calcium-binding protein, known to interact with Munc13 to promote exocytosis; whether it plays a role in agonist-regulated exocytosis is unknown. To address this question, we isolated chromaffin cells from Doc2B (+/+), (+/-) and (-/-) mice and examined the effects of PLC-coupled GPCR activation on depolarization-evoked exocytosis.We found that under control stimulus conditions, depolarization-evoked exocytosis, measured as changes in membrane capacitance, was not significantly different between Doc2B (+/+), (+/-) and (-/-) cells; similarly no differences were found between the sizes of the releasable vesicle pools (IRP, RRP, SRP). Equimolar replacement of extracellular Ca2+ by Ba2+ also revealed no significant differences in the synchronous and asynchronous phases of Ba2+-evoked exocytosis in Doc2B (+/+), (+/-) or (-/-) cells. Finally, although more than 80% of cells, independent of their genotype, responded to histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and, even in Doc2 (+/+) cells rarely observed (5/17 cells, (+/-): 5/24 cells, (-/-): 2/18). This is in stark contrast to bovine cells, where 80% of the cells show a 5-fold potentiation of exocytosis. Importantly, Doc2B (+/+), (+/-) and (-/-) cells responded to the DAG analog PMA with potentiation. Taken together, the results suggest that there are profound species differences in the coupling between GqPCRs and the exocytotic machinery in chromaffin cells and that Doc2B is not required for agonist-dependent potentiation of exocytosis in mice.

Involvement of Synaptic Protein Munc18 in the Process of Release of Catecholamines by Chromaffin Cells of the Rat Adrenal Gland

We studied the role of one of the synaptic proteins belonging to the SM-protein family, Munc18, in the process of exocytosis. For this purpose, we used microinjections of antibodies against protein Munc18 (anti- Munc18) into isolated chromaffin cells of the rat adrenal gland, with the aim of suppressing the function of this protein. Secretion of catecholamines was measured using carbon microelectrodes and an amperometric technique. Blocking of the function of Munc18 in the studied cells led to significant suppression of the process of secretion. In this case, the frequency of secretory events decreased, and the kinetic parameters of secretory peaks changed (their amplitude decreased, and duration of the decline phase increased). The obtained data allow us to conclude that Munc18 is one of the synaptic proteins responsible for calciumdependent exocytosis by chromaffin cells and that this protein can in a specific manner influence dimensions of the fusion pores.

Down-modulation of Ca2+ channels by endogenously released ATP and opioids: from the isolated chromaffin cell to the slice of adrenal medullae.

Modifications in Ca(2+) influx may lead to profound changes in the cell activity associated with Ca(2+)-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca(2+) channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca(2+) concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.

Increased catecholamine secretion contributes to hypertension in TRPM4-deficient mice

Hypertension is an underlying risk factor for cardiovascular disease. Despite this, its pathogenesis remains unknown in most cases. Recently, the transient receptor potential (TRP) channel family was associated with the development of several cardiovascular diseases linked to hypertension. The melastatin TRP channels TRPM4 and TRPM5 have distinct properties within the TRP channel family: they form nonselective cation channels activated by intracellular calcium ions. Here we report the identification of TRPM4 proteins in endothelial cells, heart, kidney, and chromaffin cells from the adrenal gland, suggesting that they have a role in the cardiovascular system. Consistent with this hypothesis, Trpm4 gene deletion in mice altered long-term regulation of blood pressure toward hypertensive levels. No changes in locomotor activity, renin-angiotensin system function, electrolyte and fluid balance, vascular contractility, and cardiac contractility under basal conditions were observed. By contrast, inhibition of ganglionic transmission with either hexamethonium or prazosin abolished the difference in blood pressure between Trpm4-/- and wild-type mice. Strikingly, plasma epinephrine concentration as well as urinary excretion of catecholamine metabolites were substantially elevated in Trpm4-/- mice. In freshly isolated chromaffin cells, lack of TRPM4 was shown to cause markedly more acetylcholine-induced exocytotic release events, while neither cytosolic calcium concentration, size, nor density of vesicles were different. We therefore conclude that TRPM4 proteins limit catecholamine release from chromaffin cells and that this contributes to increased sympathetic tone and hypertension.

Nanocrystalline diamond microelectrode arrays fabricated on sapphire technology for high-time resolution of quantal catecholamine secretion from chromaffin cells

The quantal release of oxidizable molecules can be successfully monitored by means of polarized carbon fiber microelectrodes (CFEs) positioned in close proximity to the cell membrane. To partially overcome certain CFE limitations, mainly related to their low spatial resolution and lack of optical transparency, we developed a planar boron-doped nanocrystalline diamond (NCD) prototype, grown on a transparent sapphire wafer. Responsiveness to applied catecholamines as well as the electrochemical and optical properties of the NCD-based device were first characterized by cyclic voltammetry and optical transmittance measurements. By stimulating chromaffin cells positioned on the device with external KCl, well-resolved quantal exocytotic events could be detected either from one NCD microelectrode, or simultaneously from an array of four microelectrodes, indicating that the chip is able to monitor secretory events (amperometric spikes) from a number of isolated chromaffin cells. Spikes detected by the planar NCD device had comparable amplitudes, kinetics and vesicle diameter distributions as those measured by conventional CFEs from the same chromaffin cell.

The quantal secretion of catecholamines is impaired by the accumulation of β‐adrenoceptor antagonists into chromaffin cell vesicles

The delayed onset of certain effects of antagonists of beta-adrenoceptors (beta-blockers), such as lowering arterial blood pressure (several days), cannot be explained solely by their effects on beta-adrenoceptors, an action that occurs within minutes. Although several mechanisms have been proposed, none of them explain this temporal delay. This work aimed at providing a new explanation based on the interference of these drugs with the functional accumulation of catecholamines within neurosecretory vesicles. We used the simultaneous on-line monitoring of catecholamine and labetalol release from bovine isolated chromaffin cells and from rat perfused adrenal glands, as well as single cell amperometry, intracellular electrochemistry, patch amperometry and HPLC. Using amperometry, three beta-blockers, labetalol, atenolol and propranolol, reduced the quantal size of secretory events in chromaffin cells, accompanied by a slowing down of exocytosis. By patch amperometry, we found that treatment with beta-blockers also increases the chromaffin vesicle volume, thereby creating a functional dilution of catecholamines. Experiments with intracellular electrochemistry show that vesicles cannot uptake new catecholamines. There was progressive accumulation of labetalol in secretory vesicles of bovine adrenal chromaffin cells, and this beta-blocker was co-released with catecholamines from rat and bovine chromaffin tissues. We propose that beta-blockers are progressively concentrated into sympathetic secretory vesicles, and interfere with the storage of catecholamines and are co-released with the natural transmitters, resulting in a decrease in the sympathetic tone. This could explain the delayed onset of the hypotensive effects of beta-blockers.

Differences in the quantal release of catecholamines in chromaffin cells of rat embryos and their mothers

Studies on the bulk catecholamine release from fetal and neonatal rat adrenals, adrenal slices, or isolated chromaffin cells stimulated with high K(+), hypoxia, hypercapnia, or acidosis are available. However, a study analyzing the kinetics of quantal secretion is lacking. We report here such a study in which we compare the quantal release of catecholamines from immature rat embryo chromaffin cells (ECCs) and their mothers' (MCCs). Cell challenging with a strong depolarizing stimulus (75 mM K(+)) caused spike bursts having the following characteristics. ECCs released more multispike events and wave envelopes than MCCs. This, together with narrower single-spike events, a faster decay, and a threefold smaller quantal size suggest a faster secretory machinery in ECCs. Furthermore, with a milder stimulus (25 mM K(+)) enhanced Ca(2+) entry by L-type Ca(2+) channel activator BAY K 8644 did not change the kinetic parameters of single spikes in ECCs; in contrast, augmentation of Ca(2+) entry increased spike amplitude and width, quantal size, and decay time in MCCs. This suggests that in mature MCCs, the last exocytotic steps are more tightly regulated than in immature ECCs. Finally, we found that quantal secretion was fully controlled by L-type voltage-dependent Ca(2+) channels (VDCCs) in ECCs, whereas both L- and non-L VDCCs (N and PQ) contributed equally to secretion control in MCCs. Our results have the following physiological, pharmacological, and clinical relevance: 1) they may help to better understand the regulation of adrenal catecholamine release in response to stress during fetal life and delivery; 2) if clinically used, L-type Ca(2+) channel blockers may augment the incidence of sudden infant death syndrome (SIDS); and 3) so-called Ca(2+) promotors or activators of Ca(2+) entry through L-type VDCCs may be useful to secure a healthy catecholamine surge upon violent stress during fetal life, at birth, or to prevent the SIDS in neonates at risk.

Modulation of Adrenal Catecholamine Secretion by In Vivo Gene Transfer and Manipulation of G Protein–coupled Receptor Kinase-2 Activity

We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

Opioid receptor stimulation suppresses the adrenal medulla hypoxic response in sheep by actions on Ca(2+) and K(+) channels.

Before the preganglionic regulation of the adrenal medulla is established, hypoxia acts directly on the chromaffin cells to evoke the secretion of catecholamines. This direct action of hypoxia is suppressed by the gradual development of the preganglionic innervation and we have proposed that opioid peptides released from the adrenal splanchnic nerves may be responsible for this suppression. The effects of the specific opioid agonists DPDPE (delta-agonist), U-62066 (kappa-agonist) and DALDA (mu-agonist) on the hypoxia-evoked response were investigated in both a whole-gland preparation and in isolated adrenal chromaffin cells using amperometry, whole-cell patch clamping and measurement of cytosolic [Ca(2+)]. The combined application of mu- and kappa-type agonists abolished the hypoxia-evoked catecholamine secretion from whole perfused adrenal gland. In isolated chromaffin cells, mu- and kappa-opioid agonists reduced the rise in [Ca(2+)](i) that results from exposure to hypoxia. Both agonists decreased the voltage-dependent Ca(2+) current in these cells. The mu-agonist increased the conductance through SK-type K(+) channels and this action offset the decrease in K(+) conductance produced by exposure to hypoxia. The kappa-type agonist decreased the conductance through an action on BK-type K(+) channels, a class of channels that are not involved in initiating the direct response to hypoxia. These data suggest that opioids, through their action on SK channels and voltage-dependent Ca(2+) channels, may be responsible for the nerve-induced suppression of the hypoxic response of adrenal chromaffin cells and that these effects of endogenous opioids are mediated via mu- and kappa-type receptors.

O2-sensitive K+ channels in immortalised rat chromaffin-cell-derived MAH cells.

The regulation of K(+) channels by O(2) levels is a key link between hypoxia and neurotransmitter release in neuroendocrine cells. Here, we examined the effects of hypoxia on K(+) channels in the immortalised v-myc, adrenal-derived HNK1(+) (MAH) cell line. MAH cells possess a K(+) conductance that is sensitive to Cd(2+), iberiotoxin and apamin, and which is inhibited by ~24 % when exposed to a hypoxic perfusate (O(2) tension 20 mmHg). This conductance was attributed to high-conductance Ca(2+)-activated K(+) (BK) and small-conductance Ca(2+)-activated K(+) (SK) channels, which are major contributors to the O(2)-sensitive K(+) conductance in adrenomedullary chromaffin cells. Under low [Ca(2+)](i) conditions that prevented activation of Ca(2+)-dependent K(+) conductances, a rapidly activating and slowly inactivating K(+) conductance, sensitive to both TEA and 4-aminopyridine (4-AP), but insensitive to 100 nM charybdotoxin (CTX), was identified. This current was also reduced (by ~25 %) when exposed to hypoxia. The hypoxia-sensitive component of this current was greatly attenuated by 10 mM 4-AP, but was only slightly reduced by 10 mM TEA. This suggests the presence of delayed-rectifier O(2)-sensitive channels comprising homomultimeric Kv1.5 or heteromultimeric Kv1.5/Kv1.2 channel subunits. The presence of both Kv1.5 and Kv1.2 alpha-subunits was confirmed using immunocytochemical techniques. We also demonstrated that these K(+) channel subunits are present in neonatal rat adrenomedullary chromaffin cells in situ. These data indicate that MAH cells possess O(2)-sensitive K(+) channels with characteristics similar to those observed previously in isolated chromaffin cells, and therefore provide an excellent model for examining the cellular mechanisms of O(2) sensing in adrenomedullary chromaffin cells.

Homogeneous Ca 2+ stores in rat adrenal chromaffin cells

The localization and function of Ca 2+ stores in isolated chromaffin cells of rat adrenal medulla were investigated using confocal laser microscopy and amperometry. Binding sites for BODIPY-inositol 1,4,5-trisphosphate (IP 3), -ryanodine (Ry), and -thapsigargin (Thap) were both perinuclear and at the cell periphery. The endoplasmic reticulum (ER), which was identified by ER Tracker dye, took up fluorescent Ry and IP 3, and the majority of BODIPY-Ry-binding area was bound by fluorescent IP 3. Under Ca 2+-free conditions, the amount of caffeine-induced catecholamine secretion was 33% of that of muscarine-induced secretion, but muscarine induced little or no secretion after exposure to caffeine. Muscarine-induced Ca 2+ increases, as observed with fluo-3, lasted for a few tens of seconds under Ca 2+-free conditions, whereas a caffeine-induced Ca 2+ transient diminished rapidly with a half decay time of 3 s and this spike-like Ca 2+ transient was then followed by a sustained increase with a low level. These results indicate that IP 3 receptors and Ry receptors (RyRs) are present in common ER Ca 2+ storage and the lower potency of caffeine for secretion may be due to a rapid decrease in RyR channel activity to a low level.

Effects of vasopressin on isolated rat adrenal chromaffin cells.

It has been demonstrated that arginine vasopressin (AVP) is synthesized not only in specific hypothalamic nuclei, but also in the adrenal medulla where it is thought to regulate adrenal functions by autocrine and paracrine mechanisms. In order to further characterise the effects of AVP on rat adrenal chromaffin cells, we examined: (a) the mRNA expression for V(1a) and V(1b) AVP receptors in these cells; (b) the effects of AVP on the membrane potential and membrane currents measured with the whole-cell patch-clamp technique; and (c) effect of AVP on catecholamine release from single adrenal chromaffin cells measured with carbon fibre microelectrodes. Reverse transcription-polymerase chain reaction (RT-PCR) on tissue punch samples obtained from the adrenal medulla demonstrated message for both the V(1a) and V(1b) receptors, while material obtained from the adrenal cortex showed expression of the V(1a) receptor only. Single-cell RT-PCR conducted on acutely isolated chromaffin cells showed message for the V(1a) receptor in 84% of cells, while 38% of cells also contained message for the V(1b) receptor (n=45). Under current-clamp recording, responses to AVP application (4-40 microM) were variable; 22/34 (65%) tested cells were depolarised, 29% hyperpolarised, and the remaining cells showed a biphasic response. Changes in membrane potential of either direction were dose-dependent and accompanied by a decrease in cell membrane resistance. Under voltage-clamp (V(hold)=-60 mV), AVP evoked inward current in 27/52 (52%) and outward current in 16/52 (31%) chromaffin cells. Both types of AVP-evoked responses were blocked by co-application of a nonselective V(1a)/V(1b) antagonist. Application of AVP evoked prolonged bursts of amperometric currents (indicative of catecholamine release) in 4/9 tested cells, but reduced the currents evoked by ACh application in all tested cells (n=7). These findings demonstrate a complex action of AVP on adrenal chromaffin cells, with individual adrenal chromaffin cells responding with either excitation or inhibition. This response pattern may be related to the expression of V(1) receptor subtypes.

Modulation of ACh-induced currents in rat adrenal chromaffin cells by ligands of α 2 adrenergic and imidazoline receptors

The aim of this study was to investigate the expression of the α 2-adrenergic receptors in the adrenal medulla, and to examine the mechanism by which clonidine and related drugs inhibit acetylcholine (ACh)-induced whole-cell currents in adrenal chromaffin cells. Reverse transcription-polymerase chain reaction (RT-PCR) performed on punches of rat adrenal medulla demonstrated expression of mRNA for the α 2A-, α 2B- and α 2C-adrenergic receptors. Similar experiments conducted with tissue punches obtained from the adrenal cortex did not reveal expression of these receptor subtypes. Whole-cell currents were recorded in isolated chromaffin cells using the perforated-patch configuration. ACh (50 μM) evoked inward currents with a peak amplitude of 117.8±9.3 pA ( n=45; V hol=−60 mV). The currents were inhibited in a dose-dependent manner (0.5–50 μM) by clonidine, UK 14,304 and rilmenidine (agonists of α 2/imidazoline receptors), as well as by SKF 86466 and efaroxan (antagonists). Adrenaline and noradrenaline (50–100 μM) had no significant effect. Thus, although the adrenal medulla expresses mRNA for the α 2-adrenergic receptors, the lack of agonist–antagonist specificity observed in our whole-cell recordings (in the absence of intracellular dialysis) provides additional evidence against the possibility that these inhibitory effects are mediated by classical α 2 or imidazoline receptor interactions.

Fine structure of host‐graft relationships between transplanted chromaffin cells and CNS

Our laboratory studies have shown that transplantation of adrenal medullary tissue or isolated chromaffin cells into central nervous system (CNS) pain modulatory regions (i.e., periaqueductal gray and subarachnoid lumbar spinal cord) can reduce pain sensitivity of rats in both acute and chronic pain. The analgesia produced by these transplants is thought to result from release of both opiate peptides and catecholamines. Morphologically, these animal studies also suggest that there is no development of tolerance over long periods of time, and the transplanted chromaffin cells appear to be robust and well integrated with the host tissue. In our initial clinical studies, where allografts of adrenal medullary tissue were transplanted intrathecally to relieve intractable cancer pain, patients obtained significant and long-lasting pain relief. Increased cerebrospinal fluid (CSF) levels of metenkephalin were correlated with the decreased pain scores. Histology of autopsy tissue obtained from two patients with 1 year transplants revealed viable transplanted chromaffin cells. Because of the limited availability of human adrenal glands, sources of xenogeneic chromaffin cells will need to be identified if effective transplantation therapy for chronic pain is to be developed further.