Electrophysiology of the renin-producing juxtaglomerular cells
Ulla G. Friis,
Boye L. Jensen,
Finn Jørgensen,
Ditte Andreasen and
Ole Skøtt
Physiology and Pharmacology, University of Southern Denmark, Odense, Denmark
Correspondence and offprint requests to: Dr Ulla G. Friis, Physiology and Pharmacology, University of Southern Denmark, Winsloewparken 21,3, DK-5000 Odense C, Denmark. Email: ufriis{at}health.sdu.dk
Keywords: BKCa; Cav; cAMP; cGMP; exocytosis
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Introduction
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Renin is an aspartyl-proteinase hormone that is produced, stored and released by juxtaglomerular (JG) granular cells in the distal part of the renal afferent arterioles. The rate of secretion of renin from JG cells is one of the main determinants of the overall activity of the renin-angiotensin system, and is the most well regulated parameter of all constituents of that system.
At least three cellular messengers are involved in the intracellular control of renin secretion. Intracellular calcium is an inhibitory second messenger and is increased after exposure of the JG cells to vasoconstrictors such as angiotensin II, a1-adrenergic agonists or endothelin [1]. Cyclic AMP stimulates the secretory process and is involved in the stimulation of renin secretion seen after exposure to vasodilator hormones coupled to activation of adenylyl cyclase (dopamine [2], b-adrenergic agonists [3], prostaglandin E2, prostaglandin I2 and adrenomedullin [4]). Cyclic GMP may inhibit or stimulate renin secretion depending on which of several pathways dominate [5]. Many of the cellular and subcellular processes involved in the renin secretory control are not well understood.
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Ion channels in single JG cells
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Early electrophysiological studies using sharp electrodes on JG cells are reviewed in Friis et al. [6]. The first patch-clamp study [7] using mouse isolated glomeruli with attached afferent arterioles showed that JG cells displayed outward rectification at positive membrane potentials, and that this current was carried by K+. We applied the patch-clamp technique [8,9] to single rat JG cells to identify the contribution of the different ion channels to this membrane conductance [10]. The current-voltage (I-V) relationship was obtained by applying the pulse protocol shown in Figure 1A. An original recording of the whole-cell currents following this pulse protocol is shown in Figure 1B, and the steady-state I-V relationship is shown in Figure 1C. The cell displayed outward rectification and limited net currents between 30 mV and +10 mV [7]. Tetraethylammonium (TEA), which is an unselective blocker of potassium channels, inhibited most of the outward current suggesting that under resting conditions the outward current is carried by K+. This is in accordance with results obtained from mouse JG cells [7]. Inhibition of voltage-gated KV channels with 4-aminopyridine only slightly affected the current, indicating that only a minor part of the current is carried through KV channels. Chelation of calcium with EGTA almost completely abolished the outward current, indicating that calcium-sensitive potassium channels were involved. Consistent with this, charybdotoxin and iberiotoxin, blockers of calcium-sensitive voltage-gated (BKCa) channels, inhibited almost all of the outward current. The current was enhanced by cAMP, suggesting that the BKCa was of the cAMP-stimulated ZERO isoform. This prediction was confirmed by RTPCR [10].

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Fig. 1. Whole-cell current in a single JG cell. (A) Pulse protocol. (B) Recording of whole-cell current from a JG cell dialyzed with control solution. (C) Steady-state I-V relationship.
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The JG cells exhibited voltage-dependent calcium currents that were sensitive to the L-type calcium channel blocker calciseptine and they expressed mRNA and protein of the L-type channel Cav 1.2 [10]. A functional role of these channels was indicated by the finding that strong depolarization inhibited cAMP-mediated increases in cell membrane capacitance, but when membrane potential is not artificially clamped the action of the BKCa channels is likely to prevent depolarization in this range and, thereby, be permissive for renin release.
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The cAMP pathway
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Hormones, neurotransmitters, and autocoids that raise the intracellular production of cAMP stimulate renin secretion and renin mRNA levels [11], but exactly how cAMP affects the secretory pathways is not well understood. Exocytosis involves insertion of the renin granule membrane into the cell membrane. This leads to an increase in cell surface area and therefore to an increase in cell capacitance, which can be measured by the whole-cell patch-clamp technique [12]. We have used this technique to measure renin secretion from single JG cells [13,14]. Usually Cm was followed for at least 10 min. With control solutions the capacitance was constant over time. The average capacitance of JG cells was 3.13 pF (±0.13 pF, n = 106, mouse) and 2.82 pF (±0.06 pF, n = 192, rat).
The patch-clamp technique makes it possible to introduce molecules into the cell through the pipette and simultaneously measure changes in cell capacitance. With 1 mM cAMP in the pipette, a significant increase in Cm was observed, suggesting net addition of membrane material to the cell membrane. This observation is in accordance with the hypothesis that renin secretion occurs by exocytosis. The stable cAMP analogue, Sp-cAMPs, also increased Cm. The responses to cAMP or Sp-cAMPs were completely abolished by the protein kinase A (PKA)-blocker Rp-cAMPs, indicating that the action of cAMP is mediated via PKA. In comparison to observations in other secretory cells, the rate of increase in Cm after stimulation of JG cells was quite slow. There was a gradual increase in capacitance during the first 5 min after the whole-cell configuration was established. A similar increase in JG cell capacitance was observed after addition of the b-adrenergic agonist isoproterenol to the voltage-clamped JG cells. This is probably because of the time required to generate intracellular cAMP. We conclude from these results that receptor-mediated activation of adenylyl cyclase and subsequent formation of cAMP leads to exocytotic secretion of renin.
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The PDE-3 pathway
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Agonists coupled to cGMP formation have been reported both to stimulate and inhibit renin release [1519]. The concentrations of cyclic nucleotides in cells are determined by the rate of synthesis by cyclases and by the rate of degradation by cyclic nucleotide phosphodiesterases (PDEs) [20]. With regard to the control of renin release, the PDE3 and PDE4 subtypes have attracted special attention. Thus, PDE3- and PDE4-selective inhibitors increase renin release in conscious rabbits and humans [2124]. Similar findings have been obtained with the isolated perfused rat kidney [25]. PDE3 and PDE4 use primarily cAMP as a substrate, and PDE3 is endogenously inhibited by cGMP [20]. This raises the intriguing possibility of an interaction between hormones acting through cGMP production and the cAMP pathway in the control of renin release. Recent data from whole animal studies [21] and from the isolated kidney [25] have indeed supported the concept that cGMP-dependent agonists might enhance renin release through inhibition of PDE3. We tested this hypothesis at the cellular level, where effects on renal haemodynamics, renal nerves and signals from the macula densa are excluded [14].
First, we needed to confirm the presence of PDEs in JG cells. The expression profile for PDE3 mRNA was analysed by RTPCR, and it could be shown that PDE3A was expressed in single JG cells sampled with the patch pipette. Then the functionality of this enzyme was investigated. When the cAMP-specific PDE3 was inhibited with the PDE3-inhibitor trequinsin (105 mol/l), cAMP levels in isolated JG cells were significantly increased, confirming that PDE3 was present in JG cells and, furthermore, that it was constituently active, since its inhibition resulted in an increase in the cAMP level. Next, the effect of trequinsin on renin secretory activity was studied. Isolated JG cells were superfused and the effluent was collected with a time resolution of 2 min. In this experimental set-up, addition of trequinsin resulted in a rapid and transient stimulation of renin release to levels significantly above time controls.
Finally, we applied the patch-clamp technique to single isolated rat JG cells for measurements of cell capacitance in response to manipulations of the cAMP and cGMP pathways. With cGMP (105/l) in the patch pipette, a consistent and significant increase in Cm was observed, suggesting net addition of membrane material to the cell membrane. A similar increase in JG cell capacitance was observed after addition of the PDE3-blocker trequinsin to the voltage-clamped JG cells. The increase in Cm in response to cGMP or trequinsin was completely abolished by the PKA-blocker Rp-cAMPs, suggesting that PKA mediates the effect of cGMP (and trequinsin) on Cm in rat JG cells. The similar effects of cGMP and trequinsin on membrane capacitance suggest that both compounds act through inhibition of PDE3 leading to a subsequent increase in the cAMP formation and exocytosis.
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Conclusions
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JG cells display a characteristic I-V-relationship with marked outward rectification, which is mainly carried by potassium through calcium- and cAMP-sensitive voltage-gated BKCa channels (KCa 1.1, ZERO variant) and, to a lesser degree, through voltage-gated KV channels. The BKCa channels significantly influence the resting membrane potential. JG cells express functional L-type voltage-dependent calcium channels (Cav 1.2) whose activation can inhibit cAMP-induced renin release. Renin release induced by the cAMP pathway is exocytotic and this action of cAMP is mediated via PKA. Stimulation of renin release by cGMP involves inhibition of phosphodiesterase type 3 resulting in enhanced cAMP formation and activation of PKA.
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Acknowledgments
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This work was supported by funds from the Danish Heart Association (97-2-2-9-22527, 98-1-2-22583, 99-2-2-36-22743, 00-2-1-3-22831 and 01-1-2-30-22896); the Danish Medical Research Council (9802815, 22010159, 9902742 and 52-00-1085); Carlsbergfondet, the NOVO Nordisk Foundation; and Fonden af 17.12.1981.
Conflict of interest statement. None declared.
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Received for publication: 16. 3.05
Accepted in revised form: 5. 4.05