Rat Juxtaglomerular Cells Research Articles

Potassium currents of olfactory bulb juxtaglomerular cells: characterization, simulation, and implications for plateau potential firing

Odor identity is encoded by the activity of olfactory bulb glomeruli, which receive primary sensory input and transfer it to projection neurons. Juxtaglomerular cells (JGCs) may influence glomerular processing via firing of long lasting plateau potentials. Though inward currents have been investigated, little is known regarding potassium current contribution to JGC plateau potentials. We pursued study of these currents, with the overarching goal of creating components for a computational model of JGC plateau potential firing. In conditions minimizing calcium-activated potassium current (I(K(Ca))), we used whole cell voltage clamp and in vitro slice preparations to characterize three potassium currents in rat JGCs. The prominent component I(kt1) displayed rapid kinetics (τ(10%-90% rise), 0.6-2 ms; τ(inactivation), 5-10 ms) and was blocked by high concentration 4-aminopyridine (4-AP) (5 mM) and tetramethylammonium (TEA) (40 mM). It had half maximal activation at -10 mV (V(½)max) and little inactivation at rest. I(kt2), with slower kinetics (τ(10%-90% rise), 11-15 ms; τ(inactivation), 100-300 ms), was blocked by low concentration 4-AP (0.5 mM) and TEA (5 mM). The V(½)max was 0 mV and inactivation was also minimal at rest. Sustained current I(kt3) showed sensitivity to low concentration 4-AP and TEA, and had V(½)max of +10 mV. Further experiments, in conditions of physiologic calcium buffering, suggested that I(K(Ca)) contributed to I(kt3) with minimal effect on plateau potential evolution. We transformed these characterizations into Hodgkin-Huxley models that robustly mimicked experimental data. Further simulation demonstrated that I(kt1) would be most efficiently activated by plateau potential waveforms, predicting a critical role in shaping JGC firing. These studies demonstrated that JGCs possess a unique potassium current profile, with delayed rectifier (I(kt3)), atypical A-current (I(kt1)), and D-current (I(kt2)) in accordance with known expression patterns in olfactory bulb (OB) glomeruli. Our simulations also provide an initial framework for more integrative models of JGC plateau potential firing.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat

Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Cyclic AMP increases cytoplasmic free calcium in renin-secreting cells from rat kidney

The renin-secreting juxtaglomerular cells (JGC) in the media of the afferent arteriole at the vessel pole are the major source of circulating renin. The control of renin secretion is complex with increases in cAMP being the major stimulus and increases in intracellular free Ca2+ concentration ([Ca2+]i) being inhibitory. We measured [Ca2+]i in the afferent arteriole from mostly JGC. Manoeuvres that increase cAMP (e.g. isoproterenol) or dibutyryl-cAMP elicited an increase in [Ca2+]i which was approximately 40% of that induced by angiotensin II (3 nmol/l). The Ca2+ response occurred in 50-90% of the cases, and increasing the stimulus increased responder frequency but not response size. The response was (almost) abolished by removal of extracellular Ca2+, prevented by inhibitors of store-operated Ca2+ channels (Gd3+ and 2-aminoethoxydiphenyl-borate), but was unaffected by isradipine or protein kinase A inhibitors. It was not produced by an activator of EPACs (exchange protein activated by cAMP) and was not accompanied by changes in membrane potential. The data suggest that in rat JGC, cAMP, perhaps directly, activates store-operated Ca2+ channels to increase [Ca2+]i. One could speculate that this increase in [Ca2+]i serves to finely adjust the stimulating effect cAMP-increasing signals on the renin-angiotensin system.

ZRANB2: Structural and functional insights into a novel splicing protein

ZRANB2 was identified originally in a differential display experiment on 2-day and 10-day primary cultures of rat juxtaglomerular cells. During prolonged culture it was found to undergo down-regulation in concert with renin, the archetypical constituent of these cells. ZRANB2 has two zinc fingers that form a novel fold and show striking homology to Ran-binding protein domains. Human ZRANB2 mRNA is alternatively spliced to give two variants with different 3' ends. ZRANB2 has homologues across a range of species, the N-terminal end being particularly conserved. ZRANB2 is present in the nucleus of human cells. It binds to mRNA, as well as the essential splicing factors U170K and U2AF35 and the novel splicing component SFRS17A (formerly known as XE7). ZRANB2 is one of 20 genes up-regulated in grade III ovarian serous papillary carcinoma. Here, we review current knowledge surrounding ZRANB2.

Membrane potential and cation channels in rat juxtaglomerular cells.

The relationship between membrane potential and cation channels in juxtaglomerular (JG) cells is not well understood. Here we review electrophysiological and molecular studies of JG cells demonstrating the presence of large voltage-sensitive, calcium-activated potassium channels (BK(Ca)) of the ZERO splice variant, which is also activated by cAMP. These channels explain the hyperpolarization, which has been observed after stimulation of renin release with cAMP. In addition, there is now evidence that JG cells express functional L-type voltage-dependent calcium channels (Ca(v) 1.2), which in situations with strong depolarization lead to calcium influx and inhibition of renin release. In most in vivo situations the membrane potential is probably protected against depolarization by the BK(Ca) channels.

ATP-dependent mechanism for coordination of intercellular Ca2+ signaling and renin secretion in rat juxtaglomerular cells.

A change in intracellular Ca2+ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca2+ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca2+ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca2+ wave to up to 11.9+/-4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7+/-0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8+/-0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca2+ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P<0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca2+ signals.

Molecular and functional identification of cyclic AMP-sensitive BKCa potassium channels (ZERO variant) and L-type voltage-dependent calcium channels in single rat juxtaglomerular cells.

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca(v)) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between -60 and -10 mV. Blockade of K+ channels with TEA inhibited 83% of the current at +105 mV. Inhibition of KV channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K+ channels (BKCa) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BKCa and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BKCa was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was -32 mV and activation of BKCa with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca(v) blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca(v) 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K+-blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BKCa channels (the ZERO splice variant) and voltage-dependent L-type Ca2+ channels.

Transmural pressure control of prorenin processing and secretion in diabetic rat juxtaglomerular cells.

In diabetic patients, the elevation of plasma prorenin levels or arterial pressure is correlated with the severity of diabetic nephropathy. This study was designed to assess the effects of transmural pressure on prorenin regulation in juxtaglomerular (JG) cells from diabetes rats. The JG cells, harvested from rats intraperitoneally injected with streptozotocin 7 (early-diabetic) or 28 (late-diabetic) days previously, were exposed to atmospheric pressure (AP) and AP+40 mmHg for 12 h, and the renin secretion rate (RSR), prorenin secretion rate (PRSR), active renin content (ARC), prorenin content (PRC), and total renin content (TRC) were determined. Exposure of control JG cells to AP+40-mmHg significantly decreased RSR, PRSR, and ARC and significantly increased PRC without affecting TRC, suggesting the occurrence of pressure-mediated inhibition of prorenin processing and secretion. Exposure of early-diabetic and late-diabetic cells to AP+40-mmHg significantly decreased ARC and significantly increased PRC without affecting RSR, PRSR, or TRC. The changes in ARC and PRC were similar in the control and early-diabetic cells, but greater changes were observed in late-diabetic cells. However, when streptozotocin-treated rats were continuously treated with insulin (9 U/kg/day), the transmural pressure control of prorenin in JG cells was similar to that observed in the JG cells from control rats. In late-diabetic cells, treatment with a phospholipase C inhibitor did not alter the pressure control of ARC or PRC; however, treatment with a phospholipase D inhibitor did inhibit the changes in ARC and PRC with transmural pressure. Thus, pressure-mediated inhibition of prorenin secretion from JG cells has already been impaired in early diabetes. Pressure-induced inhibition of prorenin processing in JG cells via phospholipase D-dependent pathways is enhanced in late diabetes.

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases.

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol/L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.

Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells

The formation of the vasoactive peptide angiotensin II (AII) is dependent on the sequential action of two enzymes, renin and angiotensin converting enzyme (ACE), on the substrate angiotensinogen. Although the renin-producing cells of the kidney do not express angiotensinogen, they contain large amounts of AII in the same storage granules that contain renin. When renin expression is suppressed in these cells, AII also disappears. In the current study, we have tested whether the renin-associated disappearance of AII in renal juxtaglomerular (JG) cells is due to a renin-dependent down-regulation of granule biosynthesis and whether receptor-mediated internalization of AII could account for its concentration in these cells. Our results support a model whereby AII peptides are generated within JG cells, presumably by a mechanism which involves the action of endogenous renin on internalized, exogenous angiotensinogen.

Stimulatory action of parathyroid hormone on renin secretion in vitro: a study using isolated rat kidney, isolated rabbit glomeruli and superfused dispersed rat juxtaglomerular cells.

1. We have previously reported that pharmacological concentrations (125nmol/l) of parathyroid hormone may stimulate renin release in the stable recirculating and non-filtering isolated rat kidney. 2. In the present study we have attempted to extend these initial observations by examining the concentration-related response of renin release to parathyroid hormone, using the same model of isolated kidney, and determining whether the effect of parathyroid hormone on renin release can be demonstrated by more direct approaches. Thus, the effects of parathyroid hormone on renin secretion were investigated in two other renal preparations: isolated rabbit glomeruli and isolated rat juxtaglomerular cells. 3. In the isolated kidney, rat parathyroid hormone significantly stimulated renin accumulation in the perfusate in a concentration-related manner with a threshold of 1 nmol/l. 4. In both glomeruli and juxtaglomerular cells bovine [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide effectively and repeatedly stimulated renin release. These results imply that there is a direct stimulatory effect of parathyroid hormone on renin release. 5. We also examined the effect of [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide during extracellular calcium buffering in the glomeruli. [Nle8,18,Tyr34]parathyroid hormone-(1-34)amide was uneffective in calcium-free medium. Increasing the extracellular ionized calcium concentration to 2.5 mmol/l increased the extent of stimulation in accordance with the reported ability of parathyroid hormone to block calcium channels and relax vascular smooth muscle cells. 6. These results provide further support for the role of parathyroid hormone as a direct mediator of renin secretion; moreover, the renin-stimulating action of parathyroid hormone may be mediated through the inhibition of calcium influx.

Juxtaglomerular cells cultured on a reconstituted basement membrane.

Circulating renin levels are regulated by release from juxtaglomerular (JG) cells. Here, for the first time, we describe the primary culture of rat juxtaglomerular cells on a reconstituted basement membrane. In addition, primary cultures were transformed with a temperature-sensitive SV40 large T antigen gene to promote the development of a continuous JG cell line. Both primary cultures and transformed JG cells maintain a highly differentiated state and secrete active renin. These preparations now provide a system in which characterization of the cellular mechanisms of regulation of renin synthesis and release is possible.

A biphasic effect of noradrenaline on renin release from rat juxtaglomerular cells in vitro is mediated by alpha 1- and beta-adrenoceptors.

The direct effect of noradrenaline on renin release from juxtaglomerular (JG) cells in vitro were investigated in a dynamic superfusion system of dispersed rat renal cortical cells. At low concentrations (1-100 nmol/l), noradrenaline stimulated renin release in a dose-dependent manner, while at higher concentrations (0.1-1 mmol/l) it inhibited renin release. The stimulatory effect of 0.1 mumol noradrenaline/l was completely blocked by a beta-adrenoceptor antagonist, propranolol (0.1 mumol/l). When applied at concentrations of 1 mumol/l or 10 mumol/l, noradrenaline had no consistent effect on renin release, although 10 mumol noradrenaline/l had an inhibitory effect in the presence of propranolol (0.1 mumol/l). The inhibitory effect of noradrenaline (0.1 mmol/l) was converted to a stimulatory effect by the addition of an alpha 1-adrenoceptor antagonist (bunazosin, 1 mumol/l), but was not altered by the addition of an alpha 2-adrenoceptor antagonist (yohimbine, 1 mumol/l). These results indicate that low concentrations of noradrenaline directly stimulate renin release from JG cells by the activation of beta-adrenoceptors, while high concentrations of noradrenaline inhibit renin release by the activation of alpha 1-adrenoceptors. Accordingly, a dynamic balance may exist between beta-adrenergic stimulation and alpha 1-adrenergic depression of renin release.

Demonstration of dopamine DA-1 receptor sites in rat juxtaglomerular cells by light microscope autoradiography

The binding of the selective DA-1 receptor antagonist [3H]-SCH 23390 in sections of rat kidney was studied using combined in vitro biochemical radio-receptor assay and autoradiography. [3H]-SCH 23390 was bound to sections of rat kidney in a manner consistent with the labeling of DA-1 receptors with a dissociation constant value of 4.2 nmol/l and a Bmax value of 180.6 fmol/mg protein. Light microscope autoradiography revealed a dense accumulation of silver grains in juxtaglomerular cells and in proximal convoluted tubule cells. These findings suggest that the stimulation of renin release elicited by dopamine and DA-1 receptor agonists may be mediated by the activation of DA-1 receptors located on juxtaglomerular cells.

Rat Juxtaglomerular Cells are Endowed with DA-1 Dopamine Receptors Mediating Renin Release

Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (JG) cells prepared from rat kidneys continuously released renin into the culture medium at a rate of 17.9 +/- 1.4 ng angiotensin I/h per mg of cell proteins per 30 min (n = 14). Dopamine (1.0 microM), the DA-1 dopamine receptor agonist fenoldopam (0.5 microM), and isoproterenol (1.0 microM) increased renin secretion markedly (130-200%). Propranolol (0.1 microM) reduced the effects of isoproterenol significantly (80%), but not those of dopamine or fenoldopam. In contrast, SCH 23390 (0.01 microM), a DA-1 dopamine receptor antagonist, inhibited markedly only the renin release evoked by the latter two agonists, whereas S-sulpiride (10 microM), a DA-2 dopamine receptor antagonist, and phentolamine (10 microM), a nonselective alpha-adrenoceptor antagonist, did not modify the effects of either dopamine or fenoldopam. In rats, pithed to eliminate reflexogenic mechanisms regulating renin release, at the end of a 15 min i.v. infusion of fenoldopam (20 micrograms/kg per min) there was a significant increase in plasma renin activity. This effect was completely prevented by SCH 23390 (0.1 mg/kg i.v.) but not significantly changed by S-sulpiride (0.3 mg/kg i.v.) or phentolamine (3.0 mg/kg i.v.) plus propranolol (0.75 mg/kg i.v.). In conclusion, these results indicate that DA-1 dopamine receptors are present in rat kidney JG cells and that pharmacological stimulation of these receptors with dopamine or fenoldopam leads to renin secretion.

Guanosine 3',5'-cyclic monophosphate as a mediator of inhibition of renin release.

The role of guanosine 3',5'-cyclic monophosphate (cGMP) as an inhibitory mediator of tissue renin release was examined in two different in vitro preparations. In rat superficial cortical slices, renin release stimulated by isoproterenol (10(-5) M) was ablated by atriopeptin III (ANP, 2.1 x 10(-8) M), nitroprusside (NP, 10(-3) M), and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 10(-3) and 10(-6) M). Arachidonic acid (10(-3) M)-stimulated renin release was also inhibited by ANP and 8-BrcGMP (10(-3) and 10(-6) M). Both ANP and NP increased tissue cGMP concentrations significantly (P less than 0.05), but neither had an effect on adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. When methylene blue (10(-5) M), an inhibitor of guanylate cyclase, was added to slices incubated with isoproterenol and ANP, the inhibition of renin release by ANP was abolished. These results were confirmed in a preparation of isolated cultured rat juxtaglomerular cells. In these cells, isoproterenol induced a significant increase (58%, P less than 0.01) in renin release, which was inhibited by the addition of 8-BrcGMP (10(-6) M). These data demonstrate a direct inhibitory effect of ANP on isoproterenol- and arachidonic acid-induced renin release. The results with NP, 8-BrcGMP, and methylene blue suggest that cGMP is an intracellular mediator of this inhibition.